Methylation detection

ABSTRACT

A real-time method of detecting the presence and/or amount of a methylated or unmethylated gene of interest in a DNA-containing sample, comprises the steps of (a) contacting the DNA-containing sample with a reagent which selectively modifies unmethylated cytosine residues in the DNA to produce detectable modified residues but which does not modify methylated cytosine residues (b) amplifying at least a portion of the methylated or unmethylated gene of interest using at least one primer pair, at least one primer of which is designed to bind only to the sequence of methylated or unmethylated DNA following treatment with the reagent, wherein at least one primer in the primer pair produces a detectable fluorescence signal during amplification which is detected in real-time (c) quantifying the results of the real-time detection against a standard curve for the methylated or unmethylated gene of interest to produce an output of gene copy number.

FIELD OF THE INVENTION

The present invention is concerned with detection of epigeneticmodifications in nucleic acids. More specifically, the invention relatesto methods of detecting methylated or unmethylated forms of a gene ofinterest. The methods of the invention involve amplification techniques,in particular fluorescence based real-time and end-point PCR methods.

BACKGROUND TO THE INVENTION

Gene methylation is an important regulator of gene expression. Inparticular, methylation at cytosine residues found in CpG di-nucleotidepairs in the promoter region of specific genes can contribute to manydisease conditions through down regulation of gene expression. Forexample, aberrant methylation of tumour suppressor genes can lead todown regulation of these genes and is thus associated with the presenceand development of many cancers. Patterns of aberrant gene methylationare often specific to the tissue of origin. Accordingly, detection ofthe methylation status of specific genes is of prognostic and diagnosticutility and can be used to both determine the relative stage of adisease and also to predict response to certain types of therapy.

O6-methylguanine-DNA methyltransferase (MGMT) is a cellular DNA repairprotein that rapidly reverses, alkylation (e.g. methylation) at the O6position of guanine, thereby neutralizing the cytotoxic effects ofalkylating agents used in therapy such as temozolomide (TMZ) andcarmustine (1-3).

It has been shown that epigenetic silencing of the MGMT gene by promotersilencing shuts down gene transcription (4, 5), and reflects a commonalteration in primary human tumors that leads to MGMT deficiency (6).Epigenetic silencing of the MGMT gene has been shown to correlate withimproved survival in several studies with glioma patients treated withalkylating agent therapy (7) and has been substantiated in two clinicaltrials (8, 9). The recent randomized clinical trial suggests that theMGMT-methylation status has a predictive value for benefit from theaddition of the alkylating agent TMZ (9, 10). This finding has importantclinical implications for stratified therapy (11). While this trial hasestablished the new standard of care, for glioblastoma patients (10),the benefit of the addition of TMZ chemotherapy was heavily weighted topatients whose tumors had a methylated MGMT promoter, with 46% stillalive at 2 years, compared to only 14% of the patients with unmethylatedMGMT (9). Hence, this epigenetic alteration in tumors can now beexploited in a diagnostic test to predict benefit from alkylating agenttherapy for individualized management of patients. Beside glioblastoma,there is a published report that the MGMT methylation status may alsopredict benefit from alkylating agent containing therapy in patientswith low grade glioma, oligodendroglioma, and diffuse large B-celllymphoma (12, 13, 25)

Methylation-Specific PCR (MSP) with visualization of the results on agel (gel-based MSP assay) is widely used to determine epigeneticsilencing of genes (14), and in particular for testing MGMT promotermethylation in glioma (13, 15), although quantitative tests using othertechnologies have been developed (16-18). The nested gel-based MSP assayfor MGMT has been used to establish the predictive value of themethylation status of the MGMT gene promoter in the clinical trialsdetailed above (8, 9). This methodology is highly sensitive andaccurate, but has drawbacks for routine clinical use.

A number of fluorescence based technologies are available for real-timemonitoring of nucleic acid amplification reactions. One such technologyis described in U.S. Pat. No. 6,090,552 and EP 0912597 and iscommercially known as Amplifluor®. This method is also suitable forend-point monitoring of nucleic acid amplification reactions.

SUMMARY OF THE INVENTION

The present invention relates to an improved method of detecting thepresence/amount of a methylated or unmethylated gene of interest in aDNA-containing sample. The methods rely upon optimisation of a specificdetection technique for real time or end point detection ofamplification products in the context of methylation detection.

Accordingly, in a first aspect, the invention provides a real-timemethod of detecting the presence and/or amount of a methylated orunmethylated gene of interest in a DNA-containing sample, comprising:

-   -   (a) contacting/treating the DNA-containing sample with a reagent        which selectively modifies unmethylated cytosine residues in the        DNA to produce detectable modified residues but which does not        modify methylated cytosine residues    -   (b) amplifying at least a portion of the methylated or        unmethylated gene of interest using at least one primer pair, at        least one primer of which is designed to bind only to the        sequence of methylated or unmethylated DNA respectively        following treatment with the reagent, wherein at least one        primer in the primer pair is a primer containing a stem loop        structure carrying a donor and an acceptor moiety of a molecular        energy transfer pair arranged such that in the absence of        amplification, the acceptor moiety quenches fluorescence emitted        by the donor moiety (upon excitation) and during amplification,        the stem loop structure is disrupted so as to separate the donor        and acceptor moieties sufficiently to produce a detectable        fluorescence signal which is detected in real-time to provide an        indication of the gene copy number of the methylated or        unmethylated gene of interest    -   (c) quantifying the results of the real-time detection against a        standard curve for the methylated or unmethylated gene of        interest to produce an output of gene copy number;        characterised in that the amplification is considered valid        where the cycle threshold value is less than 40.

It can be readily envisaged that the methods of the invention areequally useful in detecting the presence/amount of methylated orunmethylated versions of a gene in a DNA-containing sample. Detection ofa methylated gene is more frequently utilised as a diagnostic indicatorand thus, this represents the preferred application of the methods ofthe invention. However, for certain genes, detection of an unmethylatedversion of the gene may be of primary relevance.

The methods of the invention allow the presence of a methylated orunmethylated gene of interest to be detected in a sample in real-time.Since the methods of the invention are quantitative methods, the(relative) amounts of the methylated or unmethylated form of the gene ofinterest can also be determined as the reaction proceeds.

The methods of the invention are applicable to detecting thepresence/amount of any gene of interest whose methylation status canusefully be determined. The methods have a range of diagnosticapplications based upon the correlation between gene methylation anddisease. Most preferably, the methylated or unmethylated gene ofinterest is MGMT. The MGMT gene encodes O6-methylguanine-DNAmethyltransferase (MGMT), which is a cellular DNA repair protein thatrapidly reverses alkylation (e.g. methylation) at the O6 position ofguanine, thereby neutralizing the cytotoxic effects of alkylating agentssuch as temozolomide (TMZ) and carmustine (1-3). MGMT is the gene symbolapproved by the HUGO Gene Nomenclature Committee. The gene is located onchromosome 10 (location 10q26) and the gene sequence is listed under theaccession numbers M29971, NM_(—)002412 and ENSG00000170430. Of course,as appropriate, the skilled person would appreciate that functionallyrelevant variants of the MGMT gene sequence may also be detectedaccording to the methods of the invention. For example, the methylationstatus of a number of splice variants may be determined according to themethods of the invention. Variant sequences preferably have at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide sequence identity with the nucleotide sequences in thedatabase entries. Computer programs for determining percentage.nucleotide sequence identity are available in the art, including theBasic Local Alignment Search Tool (BLAST) available from the NationalCenter fof Biotechnology Information.

Other preferred genes, whose methylation status can be determinedaccording to the methods of the invention, include WRN, BRCA1, PTEN andNDRG4. As shown in herein, the methylation status of these genes, toinclude variants as defined herein, can be reliably determined accordingto the methods of the invention.

WRN is the gene symbol approved by the HUGO Gene Nomenclature Committee.The gene is located on chromosome 8 (location p) and the gene sequenceis listed under the accession number ENSG00000165392. The gene encodesthe Werner syndrome associated DNA helicase from the RecQ family ofhelicases. Methylation of this gene may be linked to the incidence ofcolorectal cancer for example.

BRCA1 is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 17 (location q21-q24) andthe gene sequence is listed under the accession numbers U14680 andENSG00000012048. The gene encodes breast cancer 1, early onset which isone subunit of the BRCA1/BRCA2-containing complex. Methylation of thisgene may be linked to the incidence of breast cancer for example.

PTEN is the gene symbol approved by the HUGO Gene NomenclatureCommittee. The one is located on chromosome 10 (location q23) and thegene sequence is listed under the accession numbers U92436, NM_(—)000314and ENSG00000171862. The gene encodes phosphatase and tensin homolog(mutated in multiple advanced cancers 1). Methylation of this gene maybe linked to the incidence of a number of cancers, such as thyroidcarcinomas (Alvarez-Nunez et al., Thyroid (2006) January; 16(1):17-23),melanoma (Mirmohammadsadegh et al., Cancer Res (2006); 66:(13)6546-6552), Leukaemia (Lacayo et al., Clin Cancer Res (2006);12(16)477-4789) and gynaecological cancers such as cervical and ovariancancers (Yang et al., BMC Cancer (2006), 6:212) for example.

NDRG4 in the gene symbol approved by the HUGO Gene NomenclatureCommittee. The gene is located on chromosome 16 (location q21-q22.3) andthe can sequence is listed under the accession numbers AB044947 andENSG00000103034. The gene encodes NDRG family member 4. Methylation ofthis gene may be linked to the incidence of colorectal cancer forexample.

Real-time methods do not need to be utilised, however. Analyses can beperformed only to discover whether the target DNA is present in thesample or not. End-point amplification detection techniques utilize thesame approaches as widely used for Real Time PCR. Therefore, the methodsof the invention may encompass an end-point method of detecting thepresence and/or amount of a methylated or unmethylated gene of interestin a DNA-containing sample. Thus, the invention provides a(n end point)method of detecting the presence and/or amount of a methylated orunmethylated gene of interest in a DNA-containing sample, comprising:

-   -   (a) contacting the DNA-containing sample with a reagent which        selectively modifies unmethylated cytosine residues in the DNA        to produce detectable modified residues but which does not        modify methylated cytosine residues    -   (b) amplifying at least a portion of the methylated or        unmethylated cone of interest using at least one primer pair, at        least one primer of which is designed to bind only to the        sequence of methylated or unmethylated DNA following treatment        with the reagent, wherein at least one primer in the primer pair        is a primer containing a stem loop structure carrying a donor        and an acceptor moiety of a molecular energy transfer pair        arranged such that in the absence of amplification, the acceptor        moiety quenches fluorescence emitted by the donor moiety (upon        excitation) and during amplification, the stem loop structure is        disrupted so as to separate the donor and acceptor moieties        sufficiently to produce a detectable fluorescence signal which        is detected at the end of the amplification to provide an        indication of the gene copy number of the methylated or        unmethylated gene of interest    -   (c) quantifying the results of the detection against a standard        curve for the methylated or unmethylated gene of interest to        produce an output of gene copy number.

All embodiments of the invention are applicable to the end-point aspectsof the invention and thus apply mutatis mutandis. End point analysis mayinvoke use of a fluorescent plate reader or other suitableinstrumentation to determine the fluorescence at the end of theamplification.

It is noted that for each gene of interest, it may be possible toinvestigate methylation of the gene in as plurality of locations withinthe same gene. Thus, for example, a gene may incorporate more than oneCpG island, or multiple sites within the same CpG island may beinvestigated as appropriate. Thus, panels of genes may be investigatedin accordance with the present invention and may incorporate assessmentof multiple sites within the same gene as appropriate.

The methods of the invention are most preferably ex vivo or in vitromethods carried out on any suitable (DNA containing) test sample. In oneembodiment, however, the method may also include the step of obtainingthe sample. The test sample is a DNA-containing sample, in particular aDNA-containing sample including the gene of interest. The methods of theinvention can be used in the diagnosis of disease, in particular wheremethylation of a gene of interest is (known to be) linked to theincidence of disease. For example, methylation of a number of genes hasbeen shown in the art to be correlated with the incidence of cancer. Byselecting an appropriate gene of interest, the methods of the inventioncan thus be utilised in order to diagnose one or more cancer types.Examples include methylation of GSTPi for diagnosis of prostate cancer(for review on different prostate, markers see Sperry et al., 2006); andmethylation of GATA4 and many others for diagnosis of colorectal cancer(for review on CRC markers see Zitt et al., 2007).

The DNA-containing sample may comprise any suitable tissue sample orbody fluid. Preferably, the test sample is obtained from a humansubject. For cancer applications, the sample may comprise a tissuesample taken from the tissue suspected of being cancerous or from arepresentative bodily fluid. For example, in a preferred embodiment,where the gene of interest is MGMT, the sample may be a brain tissuesample or a cerebrospinal fluid sample. However, any other suitable testsamples in which epigenetic silencing of the MGMT gene can be determinedto indicate the presence of cancer are included within the scope of theinvention.

Other DNA-containing sample for use in the methods of the inventioninclude samples for diagnostic, prognostic, or personalised medicinaluses. These samples may be obtained from surgical samples, such asbiopsies or fine needle aspirates, from paraffin embedded tissues, fromfrozen tumor tissue samples, from fresh tumour tissue samples or from afresh or frozen body fluid, for example. Non-limiting examples includewhole blood, bone marrow, cerebrospinal fluid, peritoneal fluid, pleuralfluid, lymph fluid, serum, plasma, urine, chyle, stool, ejaculate,sputum, nipple aspirate, saliva, swabs specimens, colon wash specimensand brush specimens. The tissues and body fluids can be collected usingany suitable method, many such methods are well known in the art.Assessment of a paraffin-embedded specimen can be performed directly oron a tissue section.

“Diagnosis” is defined herein to include screening for a disease orpre-indication of a disease, identifying a disease or pre-indication ofa disease, monitoring the staging and the state and progression of thedisease, checking for recurrence of disease following treatment andmonitoring the success of a particular treatment. The methods of theinvention may also have prognostic value, and this is included withinthe definition of the term “diagnosis”. The prognostic value of themethods of the invention may be used as a marker of potentialsusceptibility to cancer or as a marker for progression from adenome tocancer. Thus patients at risk may be identified before the disease has achance to manifest itself in terms of symptoms identifiable in thepatient.

The methods of the invention may be carried out on purified orunpurified DNA-containing samples. However, in a preferred embodiment,prior to step (a) (the reagent treatment step), DNA isisolated/extracted/purified from the DNA-containing sample. Any suitableDNA isolation technique may be utilised. Examples of purificationtechniques may be found in standard texts such as Molecular Cloning—ALaboratory Manual (Third Edition), Sambrook and Russell (see inparticular Appendix 8 and Chapter 5 therein). In one preferredembodiment, purification involves alcohol precipitation of DNA.Preferred alcohols include ethanol and isopropanol. Suitable,purification techniques also include salt-based precipitation methods.Thus, in one specific embodiment the DNA purification techniquecomprises use of a high concentration of salt to precipitatecontaminants. The salt may comprise, consist essentially of or consistof potassium acetate and/or ammonium acetate for example. The method mayfurther include steps of removal of contaminants which have beenprecipitated, followed by recovery of DNA through alcohol precipitation.

In an alternative embodiment, the DNA purification technique is basedupon use of organic solvents to extract contaminants from cell lysates.Thus, in one embodiment, the method comprises use of phenol, chloroformand isoamyl alcohol to extract the DNA. Suitable conditions are employedto ensure that the contaminants are separated into the organic phase andthat DNA remains in the aqueous phase.

In preferred embodiments of these purification techniques, extracted DNAis recovered through alcohol precipitation, such as ethanol orisopropanol precipitation.

The methods of the invention may also, as appropriate, incorporate (alsoprior to step (a)) quantification or isolated/extracted/purified DNA inthe sample. Quantification of the DNA in the sample may be achievedusing any suitable means. Quantitation of nucleic acids may, forexample, be bused upon use of a spectrophotometer, a fluorometer or a UVtransilluminator. Examples of suitable techniques are described instandard texts such as Molecular Cloning—A Laboratory Manual (ThirdEdition), Sambrook and Russell (see in particular Appendix 8 therein).In a preferred embodiment, kits such as the Picogreen® dsDNAquantitation kit available, from Molecular Probes, Invitrogen may beemployed to quantify the DNA.

The methods of the invention rely upon a reagent which selectivelymodifies unmethylated cytosine residues in the DNA to produce detectablemodified residues. The reagent does not modify methylated cytosineresidues and thus allows for discrimination between unmethylated andmethylated nucleic acid molecules in a downstream process, preferablyinvolving nucleic acid amplification. The reagent may, in oneembodiment, act to selectively deaminate unmethylated cytosine residues.Thus, following exposure to the reagent the unmethylated DNA contains adifferent nucleotide sequence to that of corresponding methylated DNA.The deamination of cytosine results in a uracil residue being present,which has the same base pairing properties as thymine. Thus, theresultant sequence difference(s) may be detected in a number of ways—inthis case through use of primers which bind to the methylated orunmethylated version of the sequence (including cytosine residues oruracil residues respectively). In a preferred embodiment, the reagentwhich selectively modifies unmethylated cytosine residues in the DNA toproduce detectable modified residues but which does not modifymethylated cytosine residues comprises, consists essentially of orconsists of a bisulphite reagent (Frommer et al., Proc. Natl. Acad. Sci.USA 1992 89:1827-1831,). Several bisulphite containing reagents areknown in the art and suitable kits for carrying out the deaminationreaction are commercially available (such as the EZ DNA methylation kitfrom Zymo Research). A particularly preferred reagent for use in themethods of the invention comprises, consists essentially of or consistsof sodium bisulphite.

Once the DNA in the sample has been treated with the reagent, it is thennecessary to detect the difference in nucleotide sequence caused by thereagent. This is done using a nucleic acid amplification technique.Functionally relevant methylation is most commonly associated with thepromoter regions of genes. In particular, so called “CpG islands”include a relatively high incidence of CpG residues and are often foundin the promoter region of the gene. Thus, the methods of the inventiontypically focus on determining the presence of methylation in the CpGislands and/or promoters of the gene of interest. Various softwareprograms exist to allow CpG islands in a gene of interest to beidentified. Accordingly, the methods of the invention involve amplifyingat least a portion of the methylated or unmethylated gene of interestusing at least one primer pair. As discussed above, since the residuesof interest whose methylation status is to be investigated, aretypically found in defined CpG islands and/or in the promoter region ofthe gene of interest, the primer pair will typically amplify only aportion of the gene (in this region), rather than the entirety. Anysuitable portion of the gene may be amplified according to the methodsof the invention, provided that the amplification product is detectableas a reliable indicator of the presence of the gene of interest.Particularly readily detectable amplification products are betweenapproximately 50 and 250 bp. Even more preferably, amplification usingthe at least one primer pair for amplification of the methylated orunmethylated gene of interest produces an amplification product ofbetween approximately 100 and 200 bp. This is particularly relevant fortissue samples, especially paraffin embedded samples where limited DNAquality is typically obtained.

Short amplification products may also be advantageous in the context ofplasma and serum samples where DNA of low molecular weight ispredominant. Due to the lack of a requirement for a separate probemolecule, the methods of the invention are especially suitable fordetecting such low molecular weight DNA molecules. Thus, the methods ofthe invention may alternatively be characterised by the fact that theportion of the methylated or unmethylated gene of interest which isamplified is between 50 and 250 bp, such as between 100 and 200 bp. Theamplified product may be less than 200 bp and may be less than 150 bp,125 bp, 110 bp or 100 bp in length. Such methods may be particularlyapplicable to plasma and serum samples, but may also be useful in thecontext of preserved tissue samples (such as paraffin embedded samples).In certain embodiments, the portion of the methylated or unmethylatedgene of interest which is amplified is between approximately 50 and 200bp, such as between 75 and 125 bp or between 80 and 110 bp in length.This may be applicable to any gene of interest and in the context of anydisease indication. In certain embodiments, the gene is NDRG4. Thedisease may be colorectal cancer, where plasma and serum samples arediagnostically useful. Amplification of shorter lengths of DNA mayequally be applied to the reference gene, as discussed herein.

At least one primer in the primer pair, and preferably both primers, isdesigned to bind only to the sequence of methylated or unmethylated DNAfollowing treatment with the reagent. Thus, the primer acts todiscriminate between a methylated and an unmethylated gene by basepairing only with the either the methylated form of the gene (whichremains unmodified following treatment with the reagent) or theunmethylated form of the gene (which is modified by the reagent)depending upon the application to which the methods are put. The primermust, therefore, cover at least one methylation site in the gene ofinterest. Preferably, the primer binds to a region of the pane includingat least 1, 2, 3, 4, 5, 6, 7 or 8 methylation sites. Most preferably theprimer is designed to bind to a sequence in which all cytosine residuesin CpG pairs within the primer binding site are methylated orunmethylated—i.e. a “fully methylated” or a “fully unmethylated”sequence. However, if only a single or a raw methylation sites are offunctional relevance, the primer may be designed to bind to a targetsequence in which only these residues must be methylated (remain as acytosine) or unmethylated (converted to uracil) for effective binding totake place. Other (non-functionally relevant) potential sites ofmethylation may be avoided entirely through appropriate primer design orprimers may be designed which bind independently of the methylationstatus of these less relevant sites (for example by including a mix of Gand A residues at the appropriate location within the primer sequence).Accordingly, an amplification product is expected only if the methylatedor unmethylated form of the gene of interest was present in the originalDNA-containing sample. Additionally or alternatively, it may beappropriate for at least one primer in the primer pair to bind only tothe sequence at unmethylated DNA following treatment with the reagentand the other primer to bind to methylated DNA only followingtreatment—for example where a gene involves functionally important siteswhich are methylated and separate functionally important sites which areunmethylated.

At least one primer in the primer pair is a primer containing a stemloop or “hairpin” structure carrying a donor and an acceptor moiety of amolecular energy transfer pair. This primer may or may not be a primerwhich discriminates between methylated and unmethylated DNA as desired.The primer as arranged such that in the absence of amplification, theacceptor moiety quenches fluorescence emitted by the donor moiety uponexcitation. Thus, prior to, or in the absence of, amplification directedby the primer the stem loop or “hairpin” structure remains intact.Fluorescence emitted by the donor moiety is effectively accepted by theacceptor moiety leading to quenching of fluorescence.

During amplification, the configuration of the stem loop or hairpinstructure of the primer is altered. In particular, once the primer isincorporated into an amplification product, and in particular into adouble stranded DNA, (particularly during the second round ofamplification) the stem loop or hairpin structure is disrupted. Thisalteration in structure separates the donor and acceptor moietiessufficiently that the acceptor moiety is no longer capable ofeffectively quenching the fluorescence emitted by the donor moiety.Thus, the donor moiety produces a detectable fluorescence signal. Thissignal is detected in real-time to provide an indication of the genecopy number of the methylated or unmethylated gene of interest.

Thus, the methods of the invention utilise oligonucleotides foramplification of nucleic acids that are detectably labelled withmolecular energy transfer (MET) labels. The primers contain a donorand/or acceptor moiety of a MET pair and are incorporated into theamplified product of an amplification reaction, such that the amplifiedproduct contains both a donor and acceptor moiety of a MET pair.

When the amplified product is double stranded, the MET pair incorporatedinto the amplified product may be on the same strand or, when theamplification is triamplification, on opposite strands. In certaininstances wherein the polymerase used in amplification has 5′-3′exonuclease activity, one of the MET pair moieties may be cleaved fromat least some of the population of amplified product by this exonucleaseactivity. Such exonuclease activity is not detrimental to theamplification methods of the invention.

The methods of the invention, as discussed herein are adaptable to manymethods for amplification of nucleic acid sequences, includingpolymerase chain reaction (PCR), triamplification, and otheramplification systems.

In a preferred embodiment, the MET is fluorescence resonance energytransfer (FRET), in which the oligonucleotides are labelled with donorand acceptor moieties, wherein the donor moiety is a fluorophore and theacceptor moiety may be a fluorophore, such that fluorescent energyemitted by the donor moiety is absorbed by the acceptor moiety. Theacceptor moiety may be a quencher. Thus, the amplification primer is ahairpin primer that contains both donor and acceptor moieties, and isconfigured such that the acceptor moiety quenches the fluorescence ofthe donor. When the primer is incorporated into the amplificationproduct its configuration changes, quenching is eliminated, and thefluorescence of the donor moiety may be detected.

The methods of the invention permit detection of an amplificationproduct without prior separation of unincorporated oligonucleotides.Moreover, they allow detection of the amplification product directly, byincorporating the labelled oligonucleotide into the product.

In a preferred embodiment, the methods of the invention also involvedetermining the expression of a reference gene. Reference genes areimportant to allow comparisons to be made between different samples. Byselecting an appropriate gene believed to be expressed in a stable andreliable fashion between the samples to be compared, detectingamplification of a reference gene together with the gene of interesttakes into account inter-sample variability, such as amount of inputmaterial, enzymatic efficiency, sample degradation etc. A reference moreshould ideally, in the presence of a reliable amount of input DNA, beone which is constantly expressed between the samples under test. Thus,the results from the gene of interest can be normalised against thecorresponding copy number of the reference gene. Suitable referencegenes for the present invention include beta-actin,glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal RNA genessuch as 18S ribosomal RNA and RNA polymerase II gene (Radonic A. et al.,Biochem Biophys Res Commun. 2004 Jan. 23; 313(4):856-62). In aparticularly preferred embodiment, the reference gene is beta-actin.

Thus the methods of the invention may be further characterised inamplifying at least a portion of a reference gene using at least oneprimer pair, wherein at least one primer in the primer pair is a primercontaining a stem loop structure carrying a donor and an acceptor moietyof a molecular energy transfer pair arranged such that in the absence ofamplification, the acceptor moiety quenches fluorescence emitted by thedonor moiety (upon excitation) and during amplification, the stem loopstructure is disrupted so as to separate the donor and acceptor moietiessufficiently to produce a detectable fluorescence signal which isdetected, preferably in real-time, to provide an indication of the genecopy number of the reference gene.

Any suitable portion of the reference gene may be amplified according tothe methods of the invention, provided that the amplification product isdetectable as a reliable indicator of the presence of the referencegene. Particularly readily detectable amplification products are betweenapproximately 50 and 250 bp. Even more preferably, amplification usingthe at least one primer pair for amplification of the reference geneproduces an amplification product of between approximately 100 and 200bp. This is particularly relevant for tissue samples, especiallyparaffin embedded samples where limited DNA quality is typicallyobtained.

In the embodiments in which a reference gene is included in the methodsof the invention the methods may be further characterised in that thestep of the methods which comprises quantifying the results of the(real-time) detection against a standard curve for the methylated orunmethylated gene of interest also comprises quantifying the results ofthe real-time detection of the reference gene against a standard curvefor the reference gene, to produce an output of gene copy number in eachcase and optionally further comprises normalising the results bydividing the gene copy number of the methylated or unmethylated gene ofinterest by the gene copy number of the reference gene. Again, themethods are characterised in that the amplification is considered validwhere the cycle threshold value is leas than 40. This is preferably theare for both the gene of interest and reference gene.

Amplification of at least a portion of the reference gene generallyutilises at least one primer pair. Preferably, at least one primer inthe primer pair is a primer containing a stem loop structure carrying adonor and an acceptor moiety of a molecular energy transfer pair, as forthe gene of interest. The primer is arranged such that in the absence ofamplification, the acceptor moiety quenches fluorescence emitted by thedonor moiety upon excitation. Thus, prior to, or in the absence of,amplification directed by the primer the stem loop or “hairpin”structure remains intact. Fluorescence emitted by the donor moiety iseffectively accepted by the acceptor moiety leading to quenching offluorescence.

During amplification, the configuration of the stem loop or hairpinstructure of the primer is altered. In particular, once the primer isincorporated into an amplification product, and in particular into adouble stranded DNA, the stem loop of hairpin structure is disrupted.This alteration in structure separates the donor and acceptor moietiessufficiently that the acceptor moiety is no longer capable ofeffectively quenching the fluorescence emitted by the donor moiety.Thus, the donor moiety produces a detectable fluorescence signal. Thissignal is detected in real-time to provide an indication of the genecopy number of the reference gene. Alternatively, as discussed herein,the signal can be detected (solely) at the end-point of theamplification.

The “hairpin” primers for use in the methods of the invention are mostpreferably as described in U.S. Pat. No. 6,000,552 and EP 0912597, thedisclosures of which are hereby incorporated in their entirety. Theseprimers are commercially known as Amplifluor® primers. Thus, in aparticularly preferred embodiment, the primer containing a stem loopstructure used to amplify a portion of the gene of interest and/orreference gene comprises, consists essentially of or consists of thefollowing contiguous sequences in 5′ to 3′ order:

-   -   (a) a first nucleotide sequence of between approximately 6 and        30 nucleotides, wherein a nucleotide within said first        nucleotide sequence is labelled with a first moiety selected        from the donor moiety and the acceptor moiety of a molecular        energy transfer pair, wherein the donor moiety emits        fluorescence at one or more particular wavelengths when excited,        and the acceptor moiety absorbs and/or quenches said        fluorescence emitted by said donor moiety;    -   (b) a second, single-stranded nucleotide sequence comprising,        consisting essentially of or consisting of between approximately        3 and 20 nucleotides;    -   (c) a third nucleotide sequence comprising, consisting        essentially of or consisting of between approximately 6 and 30        nucleotides, wherein a nucleotide within said third nucleotide        sequence is labelled with a second moiety selected from said        donor moiety and said acceptor moiety, and said second moiety is        the member of said group not labelling said first nucleotide        sequence, wherein said third nucleotide sequence is        complementary in reverse order to said first nucleotide sequence        such that a duplex can form between said first nucleotide        sequence and said third nucleotide sequence such that said first        moiety and second moiety are in proximity such that, when the        donor moiety is excited and emits fluorescence, the acceptor        moiety absorbs and quenches said fluorescence emitted by said        donor moiety; and    -   (d) at the 3′ end of the primer, a fourth, single-stranded        nucleotide sequence comprising, consisting essentially of or        consisting of between approximately 8 and 40 nucleotides that        comprises at its 3′ end a sequence complementary to a portion of        the methylated or unmethylated DNA or reference gene and able to        prime synthesis by a nucleic acid polymerase of a nucleotide        sequence complementary to a nucleic acid strand comprising the        portion of the methylated or unmethylated DNA or reference gene;        wherein when said duplex is not formed, said first moiety and        said second moiety are separated by a distance that prevents        molecular energy transfer between said first and second moiety.

In a particularly preferred embodiment, the donor moiety and acceptormoiety form a fluorescence resonance energy transfer (FRET) pair.Molecular energy transfer (MET) is to process by which energy is passednon-radiatively between a donor molecule and an acceptor molecule.Fluorescence resonance energy transfer (FRET) is a form of MET. FRETarises from the properties of certain chemical compounds; when excitedby exposure to particular wavelengths of light, they emit light (i.e.,they fluoresce) at a different wavelength. Such compounds are termedfluorophores. In FRET, energy is passed non-radiatively over a longdistance (10-100 Å) between a donor molecule, which is a fluorophore,and an acceptor molecule. The donor absorbs a photon and transfers thisenergy non-radiatively to the acceptor (Förster, 1949, Z. Naturforsch,A4: 321-327; Clegg, 1992, Methods Enzymol. 211: 353-388). When twofluorophores whose excitation and emission spectra overlap are in closeproximity, excitation of one fluorophore will cause it to emit light atwavelengths that are absorbed by and that stimulate the secondfluorophore, causing it in turn to fluoresce. In other words, theexcited-state energy of the first (donor) fluorophore is transferred bya resonance induced dipole-dipole interaction to the neighbouring second(acceptor) fluorophore. As a result, the lifetime of the donor moleculeis decreased and its fluorescence is quenched, while the fluorescenceintensity of the acceptor molecule is enhanced and depolarized. When theexcited-state energy of the donor is transferred to a non-fluorophoreacceptor, the fluorescence of the donor is quenched without subsequentemission of fluorescence by the acceptor. In this case, the acceptorfunctions as a quencher. Both quenchers and acceptors may be utilised inthe present invention. Pairs of molecules that can engage influorescence resonance energy transfer (FRET) are termed FRET pairs. Inorder for energy transfer to occur, the donor and acceptor moleculesmust typically be in close proximity (up to 70 to 100 Å) (Clegg, 1992,Methods Enzymol. 211: 353-389; Selvin, 1995, Methods Enzymol. 246:300-334). The efficiency of energy transfer fails off rapidly with thedistance between the donor and acceptor molecules. According to Forster(1949, Z. Naturforsch. A4:321-327), the efficiency of energy transfer isproportional to D×10⁻⁶, where D is the distance between the donor andacceptor. Effectively, this means that FRET can most efficiently occurup to distances of about 70 Å. Molecules that are commonly used in FRETinclude fluorescein, 5-carboxyfluorescein or 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and5-(2′-aminoethyl)aminonaphthalone-1-sulfonic acid (EDANS). Whether afluorophore is a donor or an acceptor is defined by its excitation andemission spectra, and the fluorophore with which it is paired. Forexample, FAM is most efficiently excited by light with a wavelength of488 nm, and emits light with a spectrum of 500 to 650 cm, and anemission maximum of 525 nm. FAM is a suitable donor fluorophore for usewith JOE, TAMRA, and ROX (all of which have their excitation maximum at514 nm).

Thus, in one embodiment, said donor moiety and said acceptor moiety areselected from 5-carboxyfluorescein or 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX), 5-(2′-aminoethyl)aminonapthalene-1-sulfonicacid (EDANS), anthrandiamide, coumarin, terbium chelate derivatives,Malachite green, Reactive Red 4, DABCYL, tetramethyl rhodamine, pyrenebutyrate, eosine nitrotyrozine, ethidium, and Texas Red. In a furtherembodiment, said donor moiety is selected from fluorescein,5-carboxyfluorescein or 6-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Malachite green,and Reactive Red 4, and said acceptor moiety is selected from DABCYL,rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine,ethidium, and Texas Red.

In one particularly preferred embodiment, said donor moiety isfluorescein or a derivative thereof, and said acceptor moiety is DABCYL.Preferably, the fluorescein derivative comprises, consists essentiallyof or consists of 6-carboxy fluorescein.

The MET labels can be attached at any suitable point in the primers. Ina particularly preferred embodiment, the donor and acceptor moieties arepositioned on complementary nucleotides within the stem loop structure,such that whilst the stem loop is intact, the moieties are in closephysical proximity to one another. However, the primers of the inventionmay be labelled with the moieties in any position effective to allowMET/FRET between the respective donor and acceptor in the absence ofamplification and separation of the donor and acceptor once the primeris incorporated into an amplification product.

The stem loop or hairpin structure sequence does not depend upon thenucleotide sequence of the target gene (gene of interest or referencegene) since it does not bind thereto. Accordingly, “universal” stem loopor hairpin sequences may be designed which can then be combined with asequence specific primer to facilitate real-time detection of a sequenceof interest. The main sequence requirement is that the sequence forms astem loop/hairpin structure which is stable in the absence ofamplification (and thus ensures efficient quenching). Thus, the sequencespecific portion of the primer binds to a template strand and directssynthesis of the complementary strand. The primer therefore becomes partof the amplification product in the first round of amplification. Whenthe complimentary strand is synthesised, amplification occurs throughthe stem loop/hairpin structure. This separates the fluorophore andquencher molecules, thus leading to generation of fluorescence asamplification proceeds.

A particularly preferred stem loop or hairpin structure for inclusion inprimers for use in the methods of the invention comprises, consistsessentially of or consists of the nucleotide sequence:

(SEQ ID NO: 1) 5′ agcgatgcgttcgagcatcgcu

The stem loop structure is preferably found at the 5′ end of thesequence specific portion of the primer used in the amplification.

As mentioned above, this detector sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

A detailed in the experimental section, primers must be carefullyselected in order to ensure sensitivity and specificity of the methodsof the invention. Accordingly, particularly preferred primers for use indetecting methylated MGMT include a primer comprising, consistingessentially of or consisting of the nucleotide sequence set forth as:

(SEQ ID NO: 2) 5′ tttcgacgttcgtaggttttcgcand a printer comprising, consisting essentially of or consisting of thenucleotide sequence set forth as

(SEQ ID NO: 3) 5′ ctcgaaactaccaccgtcccga

Either one or both of the primers may be labelled with or synthesised toincorporate a suitable stem loop or hairpin structure carrying a donorand acceptor moiety, preferably at the 5′ end, as discussed in detailabove. In a preferred embodiment, one or both of the primer(s) islabelled with or synthesised to incorporate, preferably at the 5′ end,the stem loop structure comprising, consisting essentially of orconsisting of the nucleotide sequence set forth as SEQ ID NO: 1. Asmentioned above, this detector sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

Particularly preferred primers for use in detecting the methylationstatus of BRCA1, in particular methylated BRCA1, include a primercomprising, consisting essentially of or consisting of one of thenucleotide sequences set forth in table 9, ie SEQ ID NO:6 or SEQ IDNO:13. Primer pairs can be readily selected from the primers set forthin table 9. Further combinations may be made as appropriate.

Either one or both of the primers in each primer pair may be labelledwith or synthesised to incorporate a suitable stem loop or hairpinstructure carrying a donor and acceptor moiety, preferably at the 5′end, as discussed in detail above. In a preferred embodiment, one orboth of the primer(s) is labelled with or synthesised to incorporate,preferably at the 5′ end, the stem loop structure comprising, consistingessentially of or consisting of the nucleotide sequence set forth as SEQID NO: 1. In table 9, the detector sequence (stem loop) is found on theforward primer. However, it may equally be found on the reverse primerif desired. Thus, the forward primer may comprise, consist essentiallyof or consist of the nucleotide sequence TCGTGGTAACGGAAAAGCGC (SEQ ID NO6).

As mentioned above, the stem loop sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem icon orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

Particularly preferred primers for use in detecting the methylationstatus of WRN, in particular methylated WRN, include a primercomprising, consisting essentially of or consisting of one of thenucleotide sequences set forth in table 12, i.e. SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. Primerpairs can be readily selected from the primers net forth in table 12.Further combinations may be made as appropriate.

Either one or both of the primer in each primer pair may be labelledwith or synthesised to incorporate a suitable stem loop or hairpinstructure carrying a donor and acceptor moiety, preferably at the 5′end, as discussed in detail above. In a preferred embodiment, one orboth of the primer(s) is labelled with or synthesised to incorporate,preferably at the 5′ end, the stem loop structure comprising, consistingessentially of or consisting of the nucleotide sequence set forth as SEQID NO: 1. In table 12, the detector sequence (stem loop) is found oneither the forward or reverse primer in each primer pair. However, itmay equally be found on the other primer in each case, if desired.

As mentioned above, the stem loop sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other more is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET in effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

Particularly preferred primers for use in detecting the methylationstatus of PTEN, in particular methylated PTEN, include a primercomprising, consisting essentially of or consisting of one of thenucleotide sequences set forth in table 16, i.e. SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25. Primerpairs can be readily selected from the primers ret forth in table 16.Further combinations may be made as appropriate.

Either one or both of the primers in each respective primer pair may belabelled with or synthesised to incorporate a suitable stem loop orhairpin structure carrying a donor and acceptor moiety, preferably atthe 5′ end, as discussed in detail above. In a preferred embodiment, oneor both of the primer(s) is labelled with or synthesised to incorporate,preferably at the 5′ end, the stem loop structure, comprising,consisting essentially of or consisting of the nucleotide sequence setforth as SEQ ID NO: 1. In table 16, the detector sequence (stem loop) isfound on the forward primer. However, it may equally be found on thereverse primer if desired. Thus, the forward primer may lack the stemloop structure in this case.

As mentioned above, the stem loop sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

Particularly preferred primers for use in detecting the methylationstatus of NDRG4, in particular NDRG4, include a primer comprising,consisting essentially of or consisting of one of the nucleotidesequences set forth in table 18, i.e. SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 or SEQ IDNO:33. Primer pairs can be readily selected from the primers set forthin table 18. Further combinations may be made as appropriate.

Either one or both of the primers in each primer pair may be labelledwith or synthesised to incorporate a suitable stem loop or hairpinstructure carrying a donor and acceptor moiety, preferably at the 5′end, as discussed in detail above. In a preferred embodiment, one orboth of the primer(s) is labelled with or synthesised to incorporate,preferably at the 5′ end, the stem loop structure comprising, consistingessentially of or consisting of the nucleotide sequence set forth as SEQID NO: 1. In table 18, the detector sequence (stem loop) is found oneither the forward or reverse primer in each primer pair. However, itmay equally be found on the other primer in each case, if desired.

As mentioned above, the stein loop sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

Also provided by the present invention are primers for real timeamplification and detection of the preferred reference gene, beta-actin.Preferred primers include a primer comprising, consisting essentially ofor consisting of the nucleotide sequence set forth as:

(SEQ ID NO: 4) 5′ tagggagtatataggttggggaagtt and a primer comprising, consisting essentially of or consisting of thenucleotide sequence set forth as

(SEQ ID NO: 5) 5′ aacacacaataacaaacacaaattcac

Either one or both of the primers may be labelled with or synthesised toincorporate a suitable stem loop or hairpin structure carrying a donorand acceptor moiety, preferably at the 5′ end, as discussed in detailabove. In a preferred embodiment, one or both of the primer(s) islabelled, or synthesised to incorporate preferably at the 5′ end, withthe stem loop structure comprising, consisting essentially of orconsisting of the nucleotide sequence set forth as SEQ ID NO: 1. Asmentioned above, this detector sequence is generally labelled with aFRET pair. Preferably, one moiety in the FRET pair is found towards,near or at the 5′ end of the sequence and the other moiety is foundtowards, near or at the 3′ end of the sequence such that, when the stemloop or hairpin structure remains intact FRET is effective between thetwo moieties. In a particularly preferred embodiment, the stem loop orhairpin structure, especially the nucleic acid comprising, consistingessentially of or consisting of the sequence set forth as SEQ ID NO: 1,is labelled at the 5′ end with FAM and at the 3′ end with DABCYL. Otherpreferred combinations are discussed herein, which discussion appliesmutatis mutandis.

These primers and hairpin structures form separate aspects of thepresent invention. Further characteristics of these primers and hairpinstructures are summarized in the detailed description (experimentalpart) below. It is noted that variants of these sequences may beutilised in the present invention. In particular, additional flankingsequences may be added, for example to improve binding specificity orthe formation of a stem loop, as required. Variant sequences preferablyhave at least 90%, at least at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%nucleotide sequence identity with the nucleotide sequences of theprimers and/or probes set forth in SEQ ID NO:1 to 33 and in the relevanttables. The primers and hairpin structures may incorporate syntheticnucleotide analogues as appropriate or may be DNA, RNA or PNA based forexample, or mixtures thereof. Similarly alternative fluorescent donorand acceptor moieties/FRET pairs may be utilised as appropriate. Inaddition to being labelled with the fluorescent donor and acceptormoieties, the primers may include modified oligonucleotides and otherappending groups and labels provided that the functionality as a primerand/or stem loop/hairpin structure in the methods of the invention isnot compromised.

For each primer pair at least one primer is labelled with a donor and anacceptor moiety of a molecular energy transfer pair arranged such thatin the absence of amplification, the acceptor moiety quenchesfluorescence emitted by the donor moiety (upon excitation) and duringamplification, the stem loop structure is disrupted so as to separatethe donor and acceptor moieties sufficiently to produce a detectablefluorescence signal which is detected in real-time to provide anindication of the gene copy number of the methylated MGMT or beta-actingene. Preferably, said donor moiety and said acceptor moiety are a FRETpair. In one embodiment, said donor moiety and said acceptor moiety areselected from 5-carboxyfluorescein or 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′-tetramethyl-6-carboxyrhodamine (TAMRA),6-carboxy-X-rhodamine (ROX), 5-(2′-aminoethyl)aminonapthalene-1-sulfonicacid (EDANS), anthranilamide, coumarin, terbium chelate derivatives,Malachite green, Reactive Red 4, DABCYL, tetramethyl rhodamine, pyrenebutyrate, eosine nitrotyrosine, ethidium, and Texas Red. In a furtherembodiment, said donor moiety is selected from fluorescein,5-carboxyfluorescein or 6-carboxyfluorescein (FAM), rhodamine,5-(2′-aminoethyl)aminonapthalene-1-sulfonic acid (EDANS),anthranilamide, coumarin, terbium chelate derivatives, Malachite green,and Reactive Red 4, and said acceptor moiety is selected from DABCYL,rhodamine, tetramethyl rhodamine, pyrene butyrate, eosine nitrotyrosine,ethidium, and Texas Red. Preferably, said donor moiety is fluorescein ora derivative thereof, and said acceptor moiety is DABCYL and mostpreferably the donor moiety is 6-carboxyfluorescein. Other preferredcombinations, particularly in a multiplexing context, are discussedherein and these combinations are also envisaged for these aspects ofthe invention.

Particularly preferred primer pairs are:

Forward Primer MGMT: 5′-agcgatgcgttcgagcatcgcutttcgacgttcgtaggttttcgc (SEQ ID NO: 1 plus SEQ ID NO: 2) Reverse Primer MGMT: (SEQ ID NO: 3)5′-ctcgaaactaccaccgtcccga  Forward Primer beta-actin: 5′-agcgatgcgttcgagcatcgcutagggagtatataggttggggaa gtt (SEQ ID NO: 1 plus SEQ ID NO: 4)  Reverse Primer beta-actin:(SEQ ID NO: 5) 5′-aacacacaataacaaacacaaattcac

However, the stem loop (or another suitable stem loop/hairpin structure)may be incorporated (preferably during oligonucleotide synthesis) intothe reverse primer in each case in an alternative embodiment.

The invention also provides kits which may be used in order to carry outthe methods of the invention. The kits may incorporate any of thepreferred features mentioned in connection with the various methods (anduses) of the invention described herein. Thus, the invention provides akit for detecting the presence and/or amount of a methylated orunmethylated one of interest in a DNA-containing sample, comprising atleast one primer pair of the invention. In particular, the inventionprovides a kit for real-time detection of the (methylated) MGMT genecomprising at least one primer pair of the invention. Preferably, thekit incorporates a primer pair of the invention for detecting thepresence and/or amount of methylated MGMT and a primer pair fordetecting the presence and/or amount of a reference gene, in particularbeta-actin. Thus, the kit may comprise primer pairs comprising a primercomprising, consisting essentially of or consisting of the nucleotidesequence set forth as SEQ ID NO:2/3/7/8/9/10/11/12. The kit preferablyincludes primer pairs comprising, consisting essentially of orconsisting of the nucleotide sequence set forth as SEQ ID NOs 2 and 3since this primer pair was shown experimentally to be most effective inthe methods of the invention. Preferably, at least one primer in eachprimer pair is labelled with an appropriate stem loop or hairpinstructure to facilitate detection in real-time, as discussed above(which discussion applies here mutatis mutandis). Most preferably atleast one primer in each primer pair incorporates the stem loop orhairpin structure which comprises, consists essentially of or consistsof the nucleotide sequence set forth as SEQ ID NO:1. The stem loopstructure is labelled with an appropriate donor and acceptor moiety, asdiscussed herein (which discussion applies here mutatis mutandis).

As aforementioned, further characteristics of the primers of theinvention are summarized in the detailed description (experimental part)below. Variants of these sequences may be utilised in the presentinvention as discussed herein. Alternative fluorescent donor andacceptor moieties/FRET pairs may be utilised as appropriate, asdiscussed herein. Related kits are also envisaged for the WRN (table12—SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18or SEQ ID NO:19), BRCA1 (table 9—SEQ ID NO:6 and/or 13), PTEN (table16—SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24or SEQ ID NO:25) and NDRG4 (table 13 SEQ ID NO:26, SEQ ID NO:27, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32 or SEQ IDNO:33) genes incorporating suitable primer pairs as described herein.

In one embodiment, the kit of the invention further comprises a reagentwhich modifies unmethylated cytosine, as discussed herein. Such areagent is useful for distinguishing methylated from unmethylatedcytosine residues. In a preferred embodiment, the reagent comprisesbisulphite, preferably sodium bisulphite. This reagent is capable ofconverting unmethylated cytosine residues to uracil, whereas methylatedcytosines remain unconverted. This difference in residue may be utilisedto distinguish between methylated and unmethylated nucleic acid in adownstream process, such as PCR using primers which distinguish betweencytosine and uracil (cytosine pairs with guanine, whereas uracil pairswith adenine).

As discussed with respect to the methods of the invention herein,suitable controls may be utilised in order to act as quality control forthe methods. Accordingly, in one embodiment, the kit of the inventionfurther comprises, consists essentially of or consists of one or morecontrol nucleic acid molecules of which the methylation status is known.For the MGMT gene, these control nucleic acids may be derived from SW48cells and/or HT29 cells. These (one or more) control nucleic acidmolecules may include both nucleic acids which are known to be, ortreated so as to be, methylated and/or nucleic acid molecules which areknown to be, or treated so as to be, unmethylated. One example of asuitable internal reference gene, which is generally unmethylated, butmay be treated so as to be methylated, is beta-actin.

The kits of the invention may additionally include suitable buffers andother reagents for carrying out the claimed methods of the invention.Thus, the discussion provided in respect of the methods of the inventionapplies mutatis mutandis here and is not repeated for reasons ofconciseness. In one embodiment, the kit of the invention furthercomprises, consists essentially of, or consists or nucleic acidamplification buffers.

The kit may also additionally comprise, consist essentially of orconsist of enzymes to catalyze nucleic acid amplification. Thus, the kitmay also additionally comprise, consist essentially of or consist of asuitable polymerase for nucleic acid amplification. Examples includethose from both family A and family B type polymerases, such as Taq,Pfu, Vent etc.

The various components of the kit may be packaged separately inindividual compartments or may, for example be stored together whereappropriate.

The kit may also incorporate suitable instructions for use, which may beprinted on a separate sheet or incorporated into the kit's packaging forexample. The instructions may facilitate use of the kits of theinvention with an appropriate real-time amplification apparatus, anumber of which are commercially available.

The last step of the real-time methods of the invention involvesquantifying the results of the real-time detection against a standardcurve for the methylated or unmethylated gene of interest, andoptionally the reference gene (where included). Standard curves may begenerated using a set of standards. Each standard contains a known copynumber, or concentration, of the gene of interest and/or reference geneas appropriate. Typically, a baseline value of fluorescence will be setto account for background fluorescence. For example, in one embodimentthe Sequence Detection System (SDS) software is utilised. This softwaresets a default baseline range of cycles 3 to 15 of the amplificationreaction before amplification products are detected. A threshold valueof fluorescence is then defined at a statistically significant valueabove this baseline. Typically, the threshold is set to 10 standarddeviations above the baseline fluorescence. Appropriate software isprovided with apparatus for carrying out real-time amplificationreactions. The software automatically calculates the baseline andthreshold values for the reaction. The threshold cycle value (Ct) canthen be determined for each standard. This is the number of cyclesrequired to achieve the threshold amplification level. Thus, the greaterthe initial concentration of the gene standard in the reaction mixture,the fewer the number of cycles required to achieve, a particular yieldof amplified product. A plot of Ct against the log₁₀ of the knowninitial copy number of the set of standard DNAs produces a straightline. This is the standard curve. Thus, the Ct value for theamplification of the gene of interest and reference gene, whereutilised, can each be interpolated against the respective standard curvein order to determine the copy number in the DNA-containing sample.Thus, the output of the method is the gene copy number for each of thegene of interest and reference gene. The results may be normalised bydividing the gene copy number of the methylated or unmethylated gene ofinterest by the gene copy number of the reference gene. In a preferredembodiment, the Applied Biosystems 7900 HT fast real-time PCR system isused to carry out the methods of the invention. Preferably, SDS softwareis utilised, preferably including a suitable algorithm such as the AutoCT algorithm for automatically generating baseline and threshold valuesfor individual detectors.

As discussed in the experimental section, whilst the use of a real-timeamplification method involving primers only (i.e. does not require thepresence of a separate probe) provides certain technical advantages asdiscussed in U.S. Pat. No. 6,090,552 and EP 0912597), there is theproblem to solve of non-specific amplification. The selection ofappropriate primers helps to improve specificity and the usefulness ofthe primers can be determined by selecting an appropriate Ct value whichdefines whether the amplification should be considered a valid reaction.For the purposes of the methods of the invention, it has been found thatif the amplification is only considered valid where the cycle thresholdvalue is less than (or equal to) around 40 (such as 35 to 45, or 37 to43, or 35 to 42 or 39 to 41), sensitive and specific results can beobtained. If the threshold is only crossed after more than 40 cycles,the amplification is considered invalid and the results are notutilised.

Whilst selection of a Ct value of less than 40 for the gene of interestand optionally also for the reference gene (where included) representsthe primary validation criterion, additional validation criteria mayalso be utilised in the methods of the invention. Thus, in oneembodiment the amplification is considered valid where the slope of thestandard curve for the methylated or unmethylated gene of interest andreference gene is at least −4, indicating an amplification efficiency ofat least 77%.

In a further embodiment, the amplification is considered valid where thecoefficient of determination (R²) for at least four data points on eachcurve is above 0.990.

In a still further embodiment, the amplification is considered validwhere, in a parallel reaction using the same reagents, there is noamplification of a sample containing no DNA at the cycle threshold valueof less than 40. Any suitable sample lacking a DNA component, such as awater sample for example, may be utilised.

In a yet further embodiment, the amplification is considered validwhere, in a parallel reaction using the same reagents, there isdetectable amplification of a positive control sample known to containthe gene of interest in methylated form at the cycle threshold value ofless than 40. Any suitable positive control sample may be utilised. Asan example, where the gene of interest is MGMT the positive controlsample known to contain methylated MGMT is preferably derived from SW48cells.

A further validation criterion comprises the amplification beingconsidered valid where, in a parallel reaction using the same reagents,there is no detectable amplification of a negative control sample knownto contain the gene of interest in unmethylated form at the cyclethreshold value of less than 40. Any suitable negative control samplemay be utilised. As an example, where the gene of interest is MGMT thenegative control sample known to contain unmethylated MGMT is preferablyderived from HT29 cells.

Once the output of gene copy number has been obtained the method mayfurther comprise carrying out a statistical analysis of the resultsobtained. For example, density plots of the results obtained may begenerated to provide an indication of the distribution of results. Log₂transformation may be utilised to produce density plots and also assessthe reproducibility of the methods by a direct comparison of duplicateexperiments. Cohen's Kappa values may be calculated to provide anindication of the reproducibility of the methods (through comparison ofresults obtained through duplicate experiments or by comparing resultsobtained with the claimed methods with other known methods). ReceiverOperating Characteristics (ROC) curves may be plotted to determine thespecificity (proportion of true negative results) and sensitivity(proportion of true positive results) of the methods. The area under theROC curve (AUC) provides an indication of the overall performance of themethod. Preferably, the AUC is at least 0.90, 0.91, 0.92, 0.93, 0.94,0.95, 0,96, 0.97, 0.98 or 0.99 for the methods of the invention. In oneembodiment, the software environment R (available athttp://www.r-project.org/) is utilised to carry out the analysis.

Whilst suitable reaction conditions can be determined by one skilled inthe art, it has been found that certain primer concentrations workeffectively in the methods of the invention. Accordingly, in oneembodiment, the at least one primer pair for amplification of themethylated or unmethylated gene of interest/reference gene is used inthe amplification at a concentration of approximately 50 to 150 nM. Morepreferably, the at least one primer pair for amplification of themethylated or unmethylated gene of interest/reference gene is used inthe amplification at concentration of approximately 100 nM.

Whilst the methods of the invention may be utilised with any suitableamplification technique, it is most preferred that amplification iscarried out using the polymerase chain reaction (PCR). Thus, whilst PCRis a preferred amplification method, to include variants on the basictechnique such as nested PCR, equivalents may also be included withinthe scope of the invention. Examples include, without limitation,isothermal amplification techniques such as NASBA, 3SR, TMA andtriamplification, all of which are well known in the art and suitablereagents are commercially available. Other suitable amplificationmethods include, without limitation, the ligase chain reaction (LCR)(Barringer et al, 1990), MLPA, selective amplification of targetpolynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequenceprimed polymerase chain reaction (U.S. Pat. No 4,437,975), invadertechnology (Third Wave Technologies, Madison, Wis.), strand displacementtechnology, arbitrarily primed polymerase chain reaction (WO90/06995)and nick displacement amplification (WO2004/067726).

The real-time PCR methods of the invention generally involve steps oflowering the temperature to allow primer annealing, raising thetemperature for primer extension, raising the temperature fordenaturation and lowering the temperature for data-collection. In onespecific embodiment, the data-collection step is carried out at atemperature, of between approximately 60° C. and 64° C. most preferablyat approximately 62° C. since this has been shown to give maximallysensitive and specific results (as discussed in Example 2 below). Datacollection may be carried out at 57° C. in certain embodiments. Datacollection may be carried out for a suitable period of time, such as 30seconds or 1 minute per cycle.

Annealing temperatures may be varied as would be appreciated by oneskilled in the art, depending upon the primers utilised in the reaction.Suitable annealing temperatures herein may be between 50° C. and 90° C.Particularly useful annealing temperatures have been shown to be between55° C. and 65° C., especially 57° C. and/or 62° C. These annealingtemperatures may be used for any gene of interest, as indicated herein.

In a specific embodiment, the thermal profiling of the polymerase chainreaction comprises between 40 and 50 repeats, preferably approximately45 repeats of the cycle:

-   -   (a) approximately 50° C. for approximately 2 minutes    -   (b) approximately 95° C. for approximately 10 minutes    -   (c) approximately 95° C. for approximately 15 seconds    -   (d) approximately 62° C. for approximately 1 minute

This reaction scheme has been shown to produce specific and sensitiveresults in the methods of the invention. Alternative thermal profilesare discussed in the experimental section and may be applied asappropriate.

It is possible for the methods of the invention to be used in order todetect more than one gene of interest in the same reaction. Through theuse of several specific sets of primers, amplification of severalnucleic acid targets can be performed in the same reaction mixture. Thismay be termed “multiplexing”. In a preferred embodiment, one or bothprimers for each target may be hairpin primers labelled with afluorescent moiety and a quenching moiety that form a FRET pair.Amplification of several nucleic acid targets requires that a differentfluorescent donor and/or acceptor moiety, with a different emissionwavelength, be used to label each set of primers. During detection andanalysis after an amplification, the reaction mixture is illuminated andread at each of the specific wavelengths characteristic for each of thesets of primers used in the reaction. It can thus be determined whichspecific target DNAs in the mixture were amplified and labelled. In aspecific embodiment, two or more primer pairs for amplification ofdifferent respective target sequences are used. Thus the presence and/oramount of a panel of methylated/unmethylated genes of interest can bedetected in a single DNA-containing sample

Multiplexing can also be utilised in the context of detecting both thegene of interest and a reference gene in the same reaction. Again,primers labelled with appropriate his donor and/or acceptor moietiesallow the signal generated by amplification of the gene of interest andreference gene respectively to be distinguished.

In one embodiment, a universal quencher is utilised together withsuitable fluorophore donors each having a distinguishable emissionwavelength maximum. A particularly preferred quencher is DABCYL.Together with DABCYL as quencher, the following fluorophores may each beutilised to allow multiplexing: Coumarin (emission maximum of 475 nm),EDANS (491 nm), fluorescein (515 nm), Lucifer yellow (523 nm), BODIPY(525 nm), Eosine (543 nm), tetramethylrhodamine (575 nm) and texas red(615 nm) (Tyagi et al., Nature Biotechnology, Vol. 16, January 1998;49-53). Other preferred combinations are discussed herein.

In an alternative embodiment, the DNA-containing sample can be split andthe methods of the invention carried out on suitable portions of thesample in order to obtain directly comparable results. Thus, where boththe gene of interest and a reference gene are detected, the sample maybe split two was to allow detection of amplification of the gene ofinterest in real time in one sample portion and detection ofamplification of the reference gene in real time in the other sampleportion. The sample may be split further to allow suitable controlreactions to be carried out, as required. The benefit of this scheme isthat a universal FRET pair can be used to label each primer pair andremoves the requirement to detect emission at a range of wavelengths.However, this method does rely upon obtaining a suitable sampleinitially to permit dividing the sample. Whilst any suitable reactionvolume may be utilised, in one specific embodiment, the total reactionvolume for the amplification step is between approximately 10 and 40 μl,more preferably between approximately 20 and 30 μl and most preferablyaround 25 μl.

As mentioned above, epigenetic silencing of the MGMT gene has been shownto correlate with improved survival in several studies with gliomapatients treated with alkylating agent therapy (7) and has beensubstantiated in two clinical trials (8, 9). The methylation status ofMGMT is believed to have a predictive value for benefit from theaddition of the alkylating agent TMZ (9, 10). Hence, this epigeneticalteration in tumors can be exploited in a diagnostic test to predictbenefit from alkylating agent therapy for individualized management ofpatients. Beside glioblastoma, there is a published report that the MGMTmethylation status may also predict benefit from alkylating agentcontaining therapy in patients with low grade glioma, oligodendroglioma,and diffuse large B-cell lymphoma.

Accordingly, in a further aspect the invention provides a method ofpredicting the likelihood of successful treatment of a cellproliferative disorder in a subject using an alkylating chemotherapeuticagent comprising, in a DNA-containing sample isolated from the subject,detecting the presence and/or amount of methylated MGMT gene in thesample by carrying out a method of the invention, wherein the presenceof methylated MGMT in the sample indicates that the likelihood ofsuccessful treatment using the alkylating chemotherapeutic agent ishigher than if no or lower levels of methylated MGMT is detected. Ofcourse, the reverse situation is also applicable and so the methods ofthe invention may likewise be utilised in order to determine whetherthere is likely to be resistance to, or unsuccessful treatment using, analkylating chemotherapeutic agent—the absence of methylated MGMT in thesample indicates there is likely to be resistance to treatment and/orthat treatment is likely to be unsuccessful. Thus, the methods of theinvention may also be utilised to select a suitable, course of treatmentfor a patient—the presence of methylated MGMT indicates alkylatingagents may be beneficially administered, whereas the absence or lowlevel of methylated MGMT indicates alkylating agents arecontra-indicated. The discussion provided in respect of the methods,primers and kits of the invention applies to the present aspect mutatismutandis and all embodiments are therefore envisaged, as appropriate,for this aspect of the invention.

In a preferred embodiment, the cell proliferative, disorder is cancer.Preferably, the cancer is cancer of the central nervous system andespecially the brain. In a specific embodiment, the cancer of the braincomprises, consists essentially of or consists of a glioma. Preferably,the glioma comprises, consists essentially of or consists of aglioblastoma or oligodendroglioma. In an alternative embodiment, thecell proliferative disorder is diffuse large B-cell lymphoma.

The alkylating agent may be any suitable agent useful for treating acellular proliferative disorder. Preferably, the alkylatingchemotherapeutic agent is selected from carmustine, lomustine,cisplatin, carboplatin, mechlorethamine, cyclophosphamide, ifosfamide,melphalan, chlorambucil, busulfan, thiotepa, dacarbazine, temozolamideor procarbazine. All of these agents are known in the art.

Methylation of MGMT, WRN, BRCA1, PTEN and NDRG4 appears to be linked tocertain cancer types. Accordingly, in a specific embodiment, theinvention provides a method of detecting a predisposition to, or theincidence of, cancer of the central nervous system and especially thebrain or diffuse large B cell lymphoma in a sample comprising detectingmethylation of the MGMT gene using the methods of the invention, whereindetection of methylation is indicative of a predisposition to, or theincidence of, cancer and in particular cancer of the central nervoussystem and especially the brain or diffuse large B cell lymphoma.Corresponding methods are envisaged for WRN and colorectal cancer, BRCA1and breast cancer, NDRG4 and colorectal cancer and PTEN and cancers suchas thyroid carcinomas, melanoma, Leukaemia and gynaecological cancerssuch as cervical and ovarian cancers.

In a related embodiment, the invention provides a method for determiningthe histopathological stage of cancer of the central nervous system andespecially the brain or diffuse large cell lymphoma in a samplecomprising detecting methylation of the MGMT gene using the methods ofthe invention, wherein detection of methylation is indicative of thehistopathological stage of the cancer of the central nervous system andespecially the brain or diffuse large cell carcinoma. All embodiments ofthe methods of the invention are hereby incorporated as appropriate andare not repeated for reasons of conciseness. The “stage” of a cancer isa descriptor (usually numbers I to IV) of how much the cancer hasspread. The stage often takes into account the size of a tumour, howdeep it has penetrated, whether it has invaded adjacent organs, if andhow many lymph nodes it has metastasized to, and whether it has spreadto distant organs. Staging of cancer is important because the stage atdiagnosis is the biggest predictor of survival, and treatments are oftenchanged based on the stage. Again, corresponding methods are envisagedfor WRN and colorectal cancer, BRCA1 and breast cancer, NDRG4 andcolorectal cancer and PTEN and cancers such as thyroid carcinomas,melanoma, Leukaemia and gynaecological cancers such as cervical andovarian cancers.

Testing can be performed diagnostically or in conjunction with atherapeutic regimen. Epigenetic loss of function of the MGMT gene can berescued by the use of DNA demethylating agents and/or DNAmethyltransferase inhibitors. Testing can be used to determine whattherapeutic or preventive regimen to employ on a patient and be used tomonitor efficacy of a therapeutic regimen.

Accordingly, in one embodiment, the methods of the invention are appliedin a method for predicting the likelihood of successful treatment ofcancer of the central nervous system and especially the brain or diffuselarge B cell lymphoma with a DNA demethylating agent and/or a DNAmethyltransferase inhibitor and/or HDAC inhibitor comprising detectingmethylation of the MGMT gene using the methods of the invention, whereindetection of methylation is indicative that the likelihood of successfultreatment is higher than if the epigenetic modification is not detected.

In an opposite scenario, the invention provides for a method forpredicting the likelihood of resistance to treatment of cancer of thecentral nervous system and especially the brain or diffuse large B celllymphoma with a DNA demethylating agent and/or a DNA methyltransferaseinhibitor and/or HDAC inhibitor comprising detecting methylation of theMGMT gene using the methods of the invention, wherein detection ofmethylation is indicative that the likelihood of resistance to treatmentis lower than if the epigenetic modification is not detected.

Thus, the patient population may be selected for treatment on the basisof their methylation status with respect to the MGMT gene. This leads toa much more focussed and personalised form of medicine and thus leads toimproved success rates since patients will be treated with drugs whichare most likely to be effective.

Once again, corresponding methods are envisaged for WRN and colorectalcancer, BRCA1 and breast cancer, NDRG4 and colorectal cancer and PTENand cancers such as thyroid carcinomas, melanoma, Leukaemia andgynaecological cancers such as cervical and ovarian cancers.

The invention further provides for a method of selecting a suitabletreatment regimen for cancer of the central nervous system andespecially the brain or diffuse large B cell lymphoma comprisingdetecting methylation of the MGMT gene using the methods of theinvention, wherein detection of methylation results in selection of aDNA demethylating agent and/or a DNA methyltransferase inhibitor and/ora HDAC inhibitor for treatment and wherein if methylation is notdetected, a DNA demethylating agent end/or a DNA methyltransferaseinhibitor and/or a HDAC inhibitor is not selected for treatment. In theevent that methylation is not detected, alternative treatments should beexplored.

In another aspect, the invention provides for a method of treatingcancer and in particular cancer of the central nervous system andespecially the brain or diffuse large B cell lymphoma comprisingadministration of a DNA demethylating agent and/or a HDAC inhibitorand/or a DNA methyltransferase inhibitor wherein the subject has beenselected for treatment using the methods of the invention.

Thus, for the patient population where the MGMT gene is methylated,which leads to decreased gene expression, this type of treatment isrecommended.

In a related aspect, the invention also provides for the use of a DNAdemethylating agent and/or a DNA methyltransferase inhibitor and/or HDACinhibitor (in the manufacture of a medicament for use) in treatingcancer of the central nervous system and especially the brain or diffuselarge cell lymphoma in a subject, wherein the subject has been selectedfor treatment on the basis of the methods of the invention.

Once again, corresponding methods are envisaged for WRN and colorectalcancer, BRCA1 and breast cancer, NDRG4 and colorectal cancer and PTENand cancers such as thyroid carcinomas, melanoma, Leukaemia andgynaecological cancers such as cervical and ovarian cancers.

For all of the relevant methods (pharmacogenetic methods, treatmentregimen methods and methods of treatment) of the invention, the DNAdemethylating agent may be any agent capable of up regulatingtranscription of the appropriate gene. A preferred DNA demethylatingagent comprises, consists essentially of or consists of a DNAmethyltransferase inhibitor. The DNA methyltransferase inhibitor may beany suitable inhibitor of DNA methyltransferase which is suitable fortreating cancer in the presence of methylation of the relevant gene. Thelink between gene methylation and a respective cancer type is known forthese genes and so preventing this methylation is predicted to help totreat cancer.

The DNA methyltransferase inhibitor may, in one embodiment, be one whichreduces expression of DNMT genes, such as suitable antisense molecules,or siRNA molecules which mediate RNAi for example. The design of asuitable siRNA molecule is within the capability of the skilled personand suitable molecules can be made to order by commercial entities (suchas Ambion (www.ambion.com)). Preferably, the DNA methyltransferase geneis (human) DNMT1.

Alternatively, the agent may be a direct inhibitor of DNMTs. Examplesinclude modified nucleotides such as phosphorothioate modifiedoligonucleotides (see FIG. 6 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31) and nucleosides andnucleotides such as cytidine analogues. Suitable examples of cytidineanalogues include 5-azacytidine, 5-aza-2′-deoxycytidine,5-fluouro-2′-deoxycytidine, pseudoisocytidine,5,6-dihydro-5-azacytidine, 1-β-D-arabinofuranosyl-5-azacytosine (knownas fazabarine) (see FIG. 4 of Villar-Garea, A. And Esteller, M. DNAdemethylating agents and chromatin-remodelling drugs: which, how andwhy? Current Drug Metabolism, 2003, 4, 11-31).

In another embodiment, the DNA methyltransferase inhibitor comprisesDecitabine. Full details of this drug can be found at www.supergen.comfor example.

Additional DNMT inhibitors include S-Adenosyl-Methionine (SAM) relatedcompounds like ethyl group donors such as L-ethionine and non-alkylatingagents such as S-adenosyl-homocysteine (SAH), sinefungin,(S)-6-methyl-6-deaminosinefungin, 6-deaminosinefungin,N4-adenosyl-N4-methyl-2,4-diaminobutanoic acid,5′-methylthio-5′-deoxyadenosine (MTA) and 5′-amino-5′-deoxyadenosine(Villar-Garea, A. And Esteller, M. DNA demethylating agents andchromatin-remodelling drugs: which, how and why? Current DrugMetabolism, 2003, 4, 11-31).

Further agents which may alter DNA methylation and which may, therefore,be useful in the present compositions include organohalogenatedcompounds such as chloroform etc, procianamide, intercalating agentssuch as mitomycin C, 4-aminobiphenyl etc, inorganic salts of arsenic andselenium and antibiotics such as kanamycin, hygromycin and cefotaxim(Villar-Garea, A. And Esteller, M. DNA demethylating agents andchromatin-remodelling drugs: which, how and why? Current DrugMetabolism, 2003, 4, 11-31).

Particularly preferred DNMT inhibitors in the present inventioncomprise, consists essentially of or consists of 5-azacytidine and/orzebulaine.

The histone deacetylase inhibitor may comprise at least one oftrichostatin A (TSA), suberoyl hydroxamic acid (SBHA),6-(3-chlorophenylureido)caproic hydroxamic acid (3-C1-UCHA),m-carboxycinnamic acid bishydroxylamide (CBHA), suberoylanilidehydroxamic acid (SAHA), azelaic bishydroxamic acid (ABHA), pyroxamide,scriptaid, aromatic sulfonamides bearing a hydroxamic acid group,oxamflatin, trapoxin, cyclic-hydroxamic-acid containing peptides,FR901225, MS-275, MGCD0103, short-chain fatty acids andN-acetyldinaline.

The invention will now be described with respect to the followingnon-limiting examples:

DETAILED DESCRIPTION OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Decision tree for determination of the MGMT methylation statusin clinical samples (real-time MSP).

FIG. 2: Reproducibility of real-time MSP duplicate measurements. Dottedline represents identity line (x=y), and dashed lines represent thecut-off between m_MGMT and non-m_MGMT samples according to Gaussianmixture model as defined. Pearson correlation 0.996, Spearmancorrelation 0.93, N=99. Black dots represent samples with m_MGMTcopies>20; Grey star represent samples with less than 20 m_MGMT copiesbut with a m_MGMT Ct value<40.

FIG. 3: Density plot of normalized m_MGMT copy number in glioblastoma.Histogram of average results from 99 samples with duplicatemeasurements, the respective lines represent results from eachreplicate. Only samples with Ct values<40 for m_MGMT are included. Theminimum between the two Gaussian curves is at the ratio of 3log₂(m_MGMT/ACTB*1000).

FIG. 4A, FIG. 4B, FIG. 4C, and FIG. 4D: Definition of natural cut-offfor m_MGMT. Density (FIG. 4A), Classification (FIG. 4B), Uncertainty(FIG. 4C) of observations belonging to classes and posterior Probability(FIG. 4D) to be in class 2 (m_MGMT) from a fitted mixture model appliedto the average log₂(m_MGMT/ACTB*1000). In the classification plot (FIG.4B), all of the data are displayed at the bottom, with the separatedclasses shown at different levels above. The dashed line represents theoptimal cut-off according to the selected model (3 in log₂ units, orratio of 8). The area between the grey dotted lines (FIG. 4D) defines apossible grey zone; the threshold for 95% probability of non-m_MGMT is 2in log₂ space (ratio of 4), and of 4 in log₂ space (ratio of 16) form_MGMT, respectively.

FIG. 5. Comparison between direct real-time MSP and nested, gel-basedMSP results. Boxplots and stripcharts of the log₂(m_MGMT/ACTB*1000)(y-axis) of the results determined for 91 samples by direct real-timeMSP assay are compared to the respective classification by the nested,gel-based assay into non-m_MGMT and m_MGMT (M) samples (x-axis). Dashedlines represent the cut-off between m_MGMT and non-m_MGMT samples as toGaussian mixture model.

EXAMPLE 1 Correlation Between Testing MGMT Gene Promoter MethylationStatus of Glioblastoma Tissue Using Nested Gel-Based and DirectReal-Time Fluorescence-Based Methylation Specific PCR

List of Abbreviations:

-   -   Ct values: cycles at which the amplification curves cross the        threshold value, as set automatically by the software    -   MGMT: O⁶-methylguanine-DNA methyltransferase    -   ACBT: β-actin    -   m_MGMT: methylated version of MGMT gene promoter    -   u_MGMT: unmethylated version of MGMT gene promoter    -   NTC: no-template control    -   MSP: Methylation-Specific PCR    -   ONCO: OncoMethylome Sciences    -   FDA: US Food and Drug Administration    -   RTOG: Radiation Therapy Oncology Group    -   EORTC: European Organization for Research and Treatment of        Cancer    -   FFPE: formalin fixed paraffin embedded    -   HE stain: Hematoxylin-Eosin stain    -   CHUV: Centre Hospitalier Universitaire Vaudois    -   ROC: Receiver Operating Characteristic    -   AUC: Area Under the ROC Curve    -   TMS: temozolomide

Material and Methods:

Samples and Assays:

One hundred thirty-four FFPE glioma tissues, predominantly glioblastoma,were obtained from patients who had given their informed consent.Patients were enrolled in clinical trials coordinated at the Universityhospitals in Lausanne (Switzerland), Rotterdam (The Netherlands), andRegensburg (Germany). The correlations of the MGMT status with survivalwill be part of the publications reporting the respective trials (Stuppet al.; van den Bent et al.; Hau et al.; manuscripts in preparation). Ofthese, 125 were tested once by the CHUV (gel-based MBP), 29 were testedone by ONCO (real-time MSP), and 105 were tested in duplicate by ONCO(real-time MSP). See Table, 1 for further details.

TABLE 1 Summary of the samples evaluated by both assays Duplicate SingleTotal Valid Site Samples test test tests tests [%] ONCO 134 105 (210 29239 225/239 [94.2%] tests) CHUV 125 0 125 125  94/125 [75.2%]Sample preparation:

For each tumor sample eight 5 μm consecutive sections were prepared onglass slides. An additional slide stained with Hematoxylin-Eosin (HE)was used to define the tumor area that was subsequently marked on theunstained serial sections. Only tissue comprising sufficient compact andviable tumor tissue was considered, excluding necrotic areas. Forsamples with a tumor area of less than 0.5×1.0 cm the number of sectionswas increased accordingly. The prepared sample sections were thendivided between the two sites and processed in parallel at ONCO(real-time MSP) and the CHUV (gel-based MSP), according to eachlaboratory's respective protocols. The laboratories were blinded to theresults obtained in the other assay.

Nested-MSP Approach with Visualization of Results on Agarose Gels:

This assay was conducted as published previously (9). In brief, DNA wasisolated from the tumor tissue after macro dissection and scraping fromthe marked slides using the Ex-Wax DNA extraction Kit (S4530, Chemicon)limiting the proteinase digestion to six hours. After the bisulphitetreatment step, purified DNA was subjected to MCP using a two stepapproach with nested primers (19). The first round of PCR amplifies boththe methylated as well as the unmethylated version of the MGMT sequence(m_MGMT and u_MGMT). The resulting PCR product of 289 bp served as atemplate for the second, methylation-specific PCRs, amplifying eitherm_MGMT or u_MGMT, yielding PCR products of 81 and 93 bp, respectively(7). The specific primers for m_MGMT recognize the fully methylatedsequence. The u_MGMT assay served as a control for the PCR. The firstPCR consisted of 35 cycles. Two μl of the first PCR was diluted 1/20 andinput to the second PCR of 30 cycles. The products from the second PCRwere visualized on 4% agarose gels (NuSieve 3:1) to determine the MGMTmethylation status. The outcome of the nested gel-based MSP wasconsidered valid when the following four criteria were fulfilled: a) PCRproducts of the expected sizes were detected on agarose gel (singlebands); b) either a band for u_MGMT, m_MGMT, or both was present; c)routinely included positive and negative controls, including ano-template control (NTC) gave the expected result, and d) the resultwas confirmed in an independent experiment starting with the bisulphitetreatment of DNA. The valid results of all clinical samples wereclassified as methylated or non-methylated depending on the presence orabsence of a band for m_MGMT. This procedure was used for the analysisof all samples in the laboratory in the CHUV.

Real-Time MSP

DNA Isolation:

Slide sets of slices of FFPE tumor were tested using the real-time MSPassay. Of 134 samples, 29 were tested once. For 105 of the samples, theisolated DNA was split in two equal portions and the two portions wereprocessed independently beginning at the bisulphite conversion step(Table 1). Macro-dissected FFPE tissue as marked on the slides wasde-paraffinized in 750 μl xylene for 2 h. Then, 250 μl of 70% ethanolwas added before centrifugation at 13000 rpm for 15 min. The supernatantwas removed, and the samples were air dried for 20 min at roomtemperature. DNA was extracted using the classical phenol/chloroformextraction method and resuspended in 50 μl LoTe (3 mM TRIS, 0.2 mM EDTA,pH 8.0). The DNA was quantified using the Picogreen® dsDNA quantitationkit (Molecular Probes, Invitrogen) following manufacturer'srecommendations. λDNA provided with the kit was used to prepare astandard curve (from 1 to 800 ng/ml). The data were collected using aFluoStar Galaxy plate reader (BMG Lab technologies, Germany).

DNA Modification:

Up to 1.5 μq of DNA from each sample was modified using sodiumbisulphite. This reaction selectively deaminates unmethylated Cytosineresidues resulting in a conversion to Uracil, while 5-methyl Cytosineresidues are not modified. The bisulphite reaction was performed usingthe EZ DNA Modification Kit™ (Zymo, D5002) according to themanufacturer's recommendation, which includes successive steps ofconversion, desalting and desulfonation. At the end of the procedure,the modified DNA was eluted in 25 μl of 1 mM TRIS-HCL, pH 8.0, and thenstored at −80° C.

Preparation of Standards

Plasmid DNA was prepared by cloning of the relevant sequences into TOPO®TA vectors (TOPO® cloning kit, Invitrogen®). m_MGMT was obtained fromthe methylated sequence present in the SW48 cell line. After isolatingthe plasmids from the bacteria using the QIAprep® spin midiprep kit(Qiagen GmbH; according to the manufacturer's protocol), sequences wereconfirmed by MWG Biotech, Germany (data not shown). The plasmids werethen linearized by digestion with the restriction enzyme BamHI (Roche).The linearized plasmid was purified using the QIAquick® PCR purificationkit (Qiagen GmbH; according to the manufacturer's protocol). The plasmidconcentration was determined by OD₂₆₀ measurement. A stock solution of2×10⁷ copies/5 μl (4×10⁶ copies/μl) was prepared and stored at −80° C.until use. Dilutions of standard curves (2×10⁶-2×10¹ copies/5 μl) form_MGMT and ACTB were freshly prepared for each experiment.

Real-Time PCR:

The analyte (m_MGMT and ACTB) quantifications were performed byreal-time PCR assays. These consisted of parallelamplification/quantification processes using specific primer andprimer/detector pairs for each analyte using the Amplifluor® assayformat on an ABI Prism® 7900HT instrument (Applied Biosystems). Theanalyte defined in the real-time PCR was the MGMT promoter sequence anddetects the fully methylated version. ACTB was used as a reference genein the assay. The Amplifluor® forward primers are preceded by thedefection elements (underlined). The amplicon size is 136 bp for them_MGMT analyte, and 125 bp for the ACTB analyte. These amplicons includethe Amplifluor detection sequence. Sequence details for both forward andreverse primers are as follows:

Forward Primer MGT: 5′-agcgatgcgttcgagcatcgcutttcgacgttcgtaggttttc gc-3′(SEQ ID NO: 1 plus SEQ ID NO: 2) Reverse Primer MGMT: (SEQ ID NO: 3)5′-ctcgaaactaccaccgtcccga-3′ Forward Primer beta-actin:5′-agcgatgcgttcgagcatcgutagggagtatataggttgggga agtt-3′(SEQ ID NO: 1 plus SEQ ID NO: 4) Reverse Primer beta actin:(SEQ ID NO: 5) 5′-aacacacaataacaaacacaaattcac-3′

The MGMT target sequence is located on chromosome 10 between positions131155505 and 131155619, while the ACTB target sequence is located onchromosome 7 between positions 5538425 and 5538325, based on version36.1 of the NCBI human genome.

The final primer concentrations in the reaction mix were 100 nM for bothforward primer/detector and reverse primer. 12.5 μl of iTaq™ Supermixwith Rox (BioRad, 2×buffer) were used per PCR reaction. The total volumeper reaction, including 5 μl of modified template DNA, was 25 μl. TheABI 7900HT SDS instrument was started 10 min before use, allowing theheated cover to reach 105° C. The following thermal profile was used:Stage1: 50° C. for 2 min, Stage2: 95° C. for 10 min, Stage3: 95° C. for15 sec, 62° C. for 1 min (=plateau-data collection.) for 45 repeats.

Quantification:

To quantify the results of the real-time MSP assay, two standard curveswere produced, one for the reference gene (ACTB) and one for themethylated version of the MGMT gene using the standards described above.The results were generated using the SDS 2.2 software (AppliedBiosystems), exported as Ct values (cycle number at which theamplification curves cross the threshold value, set automatically by thesoftware), and then used to calculate copy numbers based on a linearregression of the standard curve values. One hundred-five clinicalsamples were measured in duplicate. Lysates of cell lines SW48 DNA andHT29 were included in each experiment as positive and negative controls,respectively, and entered the procedure at the DNA extraction step.

The results of a run were considered valid when the following fivecriteria were met: a) slopes of both standard curves above −4 (PCRefficiency>77.8%); b) R² of at least 4 relevant data points above 0.990;c) routinely included NTC not amplified; d) 10% of a 1 μq conversionreaction of the positive cell line assay control SW48 was detectable;and e) 10% of a 1 μg conversion reaction of the negative cell line assaycontrol HT29 was not detected within the standard curve.

Normalization of Results:

To compensate for variations due to differences in sample volume andpreparation, the m_MGMT copy numbers derived were divided by the ACTBcopy numbers for that sample. This figure was multiplied by 1000 forconvenient handling, and the result referred to as the ratio value.

Statistics

Statistical analyses were carried out with R, a free softwareenvironment available at http://www.r-project.org/.

Dichotomization of the Real-Time MSP Results:

The ratio values were log₂ transformed. The evaluation of thedistribution of the m_MGMT measurements in the density plot wasperformed using 99 duplicate samples (sea Table 2 for more details).Gaussian mixture models were fitted to the average datalog₂(1000*m_MGMT/ACTB) of the real-time MSP duplicates (20-22).

One sample showed a high discordance between the duplicate tests. Repeattesting of the sample produced consistent results, but this sample wasexcluded from the curve fitting exercise, which thus included 98samples.

Results

FFPE glioma tissues were analyzed in parallel to determine themethylation status of the MGMT promoter in independent laboratoriesusing two distinct technical approaches. This blinded study compared theresults obtained by a real-time MSP on a high throughput platform to thegel-based MSP assay previously shown to predict benefit from theaddition of the alkylating agent temozolomide to the treatment of newlydiagnosed glioblastoma in two clinical trials (8, 9).

Validity of Results

The assays were compared by evaluating 134 FFPE glioma samples byreal-time MSP (29 single and 105 duplicate tests) and 125 FFPE gliomasamples by gel-based MSP (all single tests as defined in references 2and 3). The real-time MSP assay produced valid results in 94.2% versus75.2% of valid tests for the gel-based assay (Table 1). Among theduplicate real-time MSP tests 5 of 6 invalid results were duplicated.The validity rates are based on the respective validation criteria foreach assay (for gel-based MSP see references 2 and 3, for real-time MSPsee FIG. 1).

Definition of Cut-Off for m_MGMT Status

Samples evaluated in duplicate by the newly established real-time MSPassay revealed a clear bimodal distribution of the measurements as shownin FIG. 3. This allows definition of a discriminatory cut-off for thedetermination of the MGMT methylation status by fitting a normal mixturemodel. The best model based on 99 samples yields 2 Gaussians of equalvariance (σ²=2.3), and of a log₂ ratio mean of −0.52 and 6.1,respectively (FIG. 4A). This corresponds to a mean ratio value[1000*m_MGMT/ACTB] of 0.7 and 68.1, respectively. According to thismodel which defines 2 classes, non-m_MGMT and m_MGMT, the cut-offcorresponds to a log₂ ratio value of 3 (FIG. 4B). Close to this cut-offthere are some samples for which the probability of belonging to oneclass or the other is close to 50% and as a direct conseouence theuncertainty of the classification is very high (FIG. 4C). This suggeststhe use of a grey zone for diagnostic purposes, and allows definition ofdifferent thresholds depending on the clinical questions asked. Thethreshold for 95% probability of methylation is a log₂ ratio value of 4,and 2 for non-m_MGMT (FIG. 4D).

Reproducibility of the Real-Time MSP Assay

Ninety nine of the 105 duplicate samples yielded a valid result for bothreal-time MSP replicates (FIG. 2). Many of the samples with very lowlog₂ ratio-values show m_MGMT copy numbers below the lower limit of thestandard curve (the lower limit of the standard curve is 20 copies). Inpractice, no ratio is calculated for these samples, and they areconsidered non-methylated. This reproducibility shows that the efficacyof the bisulphite treatment of the DNA introduced no major variabilityinto the assay.

Concordance Between Real-Time and Gel-Based MSP Assays:

The methylation status or ninety one samples with a valid result forboth real-time and gel-based MSP assays is shown in Table 2. There is agood concordance between the two tests (82/91 samples [90%]). FIG. 5shows that there is a good separation between the real-time MSP valuesfor methylated and non methylated samples. Cohen's Kappa coefficientusing a cut-off at the log₂ ratio-value of 3, was 0.80 (95% CI:0.67-0.92), similarly reflected in the one sample proportions test (withcontinuity correction) with agreement of 0/90 (95% CI: 0.82-0.95).

TABLE 2 Concordance between ONCO and CHUV results (m_MGMT and non-m_MGMTmeasurements) ONCO m_MGMT non-m_MGMT Total CHUV m_MGMT 32 1 33non-m_MGMT 8 50 58 Total 40 51 91

Discussion:

A sensitive and specific real-time MSP assay has been developed toreliably detect the methylation status of the MGMT gene promoter inclinical samples of FFPE tissues for diagnostic purposes. Themethylation status result of this test is in good concordance withresults obtained with the gel-based MSP assay that established thepredictive value of the MGMT methylation status in glioblastoma forbenefit from temozolomide therapy (9).

Although normalization provides a more reproducible assay, it maycontribute to the few discrepants seen in this study, as they are mostlysamples with detectable levels of m_MGMT but also high levels of ACTB.Some discordance is not surprising, since the cut-off of the gel-basedMSP assay is defined by visual presence or absence of the PCR productwithout normalization. Normalization to DNA content, measured here asratio to ACTB, is not an absolute measurement, since “normal tissue”contaminations are present in all clinical tumor samples, and genomiccopy number aberrations are common in glioblastoma, including onchromosomes 10 and 7, where the MGMT and ACTB gene reside, respectively.However, neither homozygous deletions nor high level amplifications havebeen reported for MGMT or ACTB that potentially could be problematic forinterpretation of results. The ACTB is also used as a control gene byother groups using real-time PCR for quantitative DNA methylationanalysis (17).

The real-time MBP assay more often yielded a valid result with FFPEtissue specimens than the gel-based MSP assay. This improved performanceis likely due to the smaller amplicon size of the real-time assay thatis more appropriate for FFPE samples known to yield limited DNA quality.

The quantitative measure obtained and the bimodal distribution of thevalues will allow definition of clinically relevant thresholds forstratified therapy. Initial cut-offs, in use today, were determined fromlimited correlation studies with the gel based MSP assay. This studyextends that correlation, confirms the initial ratio cut-offs used anddemonstrates that there is an important separation of values around thatcut-off, suggesting that there are two distinct populations underlyingthe data, and that few samples would be expected with values near thecut-off. To arrive at the best, most clinically useful cut off forpatient management, results relating the real time MSP assay to patientresponse must be studied.

The test described here is being prospectively used for randomizingpatients in an ongoing clinical phase III trial (RTOG 0525/EORTC26052-22053) testing standard versus dose-intense adjuvant TMZ inpatients with newly diagnosed glioblastoma (trial is reviewed in Stuppet al. (11)). Among the goals of this trial is to prospectively validatethe use of MGMT methylation status for predicting benefit fromalkylating agent therapy and it will provide further information on aclinically relevant cut-off for m_MGMT.

This sensitive and robust high through-put test for evaluating themethylation status of the MGMT gene may provide an importantpharmacogenomic tool for individualized management of patientsconsidered for treatment with temozolomide or other alkylating agentchemotherapy.

REFERENCES

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Mol Cell Biol 1997;        17:5612-9.    -   6. Esteller M, Hamilton S R, Burger P C, Baylin S E, Herman J G.        Inactivation of the DNA repair gene O6-methylguanine-DNA        methyltransferase by promoter hypermethylation is a common event        in primary human neoplasia. Cancer Res 1999; 59:793-7.    -   7. Esteller M, Garcia-Foncillas C, Andion E, Goodman S N,        Hidalgo O F, Vanaclocha V, et al. Inactivation of the DNA-repair        gene MGMT and the clinical response of gliomas to alkylating        agents. N Engl J Med 2000; 343:1350-4.    -   8. Hegi M E, Diserens A C, Godard S, Dietrich P Y, Regli L,        Ostermann S, et. al. Clinical trial substantiates the predictive        value, of O-6-methylguanine-DNA methyltransferase promoter        methylation in glioblastoma patients treated with temozolomide.        Clin Cancer Res 2004; 10:1871-4.    -   9. Hegi M E, Diserens A C, Gorlia T, Hamou M F, de Tribolet N,        Weller M, et al. MGMT gene silencing and benefit from        temozolomide in glioblastoma. New Engl J Med 2005; 352:997-1003.    -   10. Stupp H, Mason W P, van den Bent M J, Weller N, Fisher B,        Taphoorh M J B, et al, Radiotherapy plus concomitant and        adjuvant temozolomide for glioblastoma. N Engl J Med 2005;        352:987-96.    -   11. Stupp R, Hegi M E, van den Bent M J, Mason W R, Weller M,        Mirimanoff R O, Cairncross J G. Changing paradigms—an update on        the multidisciplinary management of malignant glioma. Oncologist        2006; 11:165-60.    -   12. Everhard S, Kaloshi C, Criniere E, Benouaich-Amiel A,        Lejeune J. Marie Y, et al. MGMT methylation: a marker of        response to temozolomide in low-grade gliomas. Ann Neural 2006;        60:740-3.    -   13. Brandes A A, Tosoni A, Cavallo G, Reni M, Franceschi E,        Bonaldi L, et al. Correlations between O6-methylguanine DNA        methyltransferase promoter methylation status, 1p and 19q        deletions, and response to temozolomide in anaplastic and        recurrent oligodendroglioma: a prospective GICNO study. J Clin        Oncol 2006; 24:4746-53.    -   14. Esteller M, Corn P G, Baylin S B, Herman J G. A gene        hypermethylation profile of human cancer. Cancer Res 2001;        61:3225-9.    -   15. Criniere E, Kaloshi G, Laigle-Donadey F, Lejeune J, Auger N,        Benoualch-Amiel A, et al. MGMT prognostic impact on glioblastoma        is dependent on therapeutic modalities. J Neurooncol 2007;        83:173-9.    -   16. Laird P W. The power and the promise of DNA methylation        markers. Nat Rev Cancer 2003; 3:253-66.    -   17. Eads C A, Danenberg K D, Kawakami K, Saltz L B, Blake C,        Shibata D, et al. MethyLight: a high-throughput assay to measure        DNA methylation. Nucleic Acids Res 2000; 28:E32.    -   18. Mikeska T, Bock C, El-Maarri O, Hubner A, Ehrentraut D,        Schramm J, et al. Optimization of Quantitative MGMT Promoter        Methylation Analysis Using Pyrosequencing and Combined Bisulfite        Restriction Analysis. J Mol Diagn 2007.    -   19. Palmisano W A, Divine K K, Saccomanno G, Gilliland F D,        Baylin S B, Herman J G, Belinsky S A. Predicting lung cancer by        detecting aberrant promoter methylation in sputum, Cancer Res        2000; 60:5954-8.    -   20. Fraley C, Raftery A E. Model-Based Clustering, Discriminant        Analysis, and Density Estimation. J American Statistical Assoc        2002; 97:611-31.    -   21. Fraley C, Raftery A E. MCLUST Version 3 for R: Normal        Mixture Modeling and Model-based Clustering. Vol. Washington:        Department of Statistics, University of Washington, 2006.    -   22. Altman D G. Practical Statistics for Medical Research.        London: Chapman & Hall, 1991.    -   23. Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner G J,        Spiegelman D, et al. Precision and performance characteristics        of bisulfite conversion and real-time PCR (MethyLight) for        quantitative DNA methylation analysis. J Mol Diagn 2006;        8:209-17.    -   24. Califice S, et al Poster 2005 “O6-Alkylguanine-DNA        Alkyltransferase Meeting”, Keele, UK, Aug. 6-9, 2005    -   25. Esteller N, Gaidano G, Goodman S N, Zagonel V, Capello D,        Botto B, Rossi D, Gloghini A, Vitolo U, Carbone A, Baylin S B,        Herman J G. Hypermethylation of the DNA repair gene        O(6)-methylguanine DNA methyltransferase and survival of        patients with diffuse large B-cell lymphoma.        -   J Natl Cancer Inst. 2002 Jan. 2; 94(1):26-32.    -   26. Sperry, et al. The emerging roles of DNA methylation in the        clinical management of prostate cancer, Endocrine-Related        Cancer (2006) 13 357-377    -   27. Zitt et al. DNA methylation in colorectal cancer—Impact on        screening and therapy monitoring modalities? Disease Markers        23 (2007) 51-71

EXAMPLE 2 Amplifluor Experiments: Testing Different Primer Sequences andReading Temperatures

Amplifluor is a primer-based methodology. It lacks the thirdoligonucleotide “probe” that typifies TaqMan and Molecular Beacontechnologies. Without this third layer of specificity, amplifluorprimers can fluoresce in response to non-specific amplification such asprimer-dimers. Therefore it is important to carefully select primersequences to overcome this behavior.

Initial Results

Initial MGMT amplifluor results were obtained using the primer set shownin Table 3. The final primer concentrations in the Amplifluor reactionmix were 100 nM for both forward primer/detector and reverse primer.12.5 μl of iTaq™ Supermix with Rox (BioRad, 2×buffer) were used per PCRreaction. The total volume per reaction, including 5 μl of no-templatecontrol (NTC: water control with no DNA present), was 25 μl. Thefollowing thermal profile was used on the ABI 7900 HT SDS instrument:Stage1: 50° C. for 2 min, Stage2: 95° C. for 10 min, Stage3: 95° C. for15 sec, 62° C. for 1 min (=plateau-data collection) for 45 repeats.

The amplifluor assay resulted in positive NTCs due to primer-dimerformation: 8 potential dimer residues were identified in this set ofprimers.

TABLE 3 primer details initial MGMT amplifluor design Name DetailsSequence 344 MGMT forward AGCGATGCGTTCGAGCATCGCUTTTCGA detectorCGTTCGTAGGTTTTCGC-3′ amplifluor detector moiety MGMTforward primer sequence (SEQ ID NO: 1 plus SEQ ID  NO: 2) PMR91MGMT reverse 5′-GCACTCTTCCGAAAACGAAACG-3′ primer (SEQ ID NO: 7)

Table 4 gives an overview of the positive Ct values observed for 12 NTCsamples tested through the initial MGMT amplifluor assay.

TABLE 4 NTC performance initial MGMT amplifluor design Ct Sample TaskPMR91 1 NTC 37.38 2 NTC 36.78 3 NTC 36.67 4 NTC 37.15 5 NTC 36.69 6 NTC36.59 7 NTC 37.17 8 NTC 36.32 9 NTC 37.71 10 NTC 36.71 11 NTC 37.86 12NTC 36.65

New Primer Designs

To overcome the problem of positive NTCs, 6 different reverse primersequences (see Table 5) were designed and 14 NTC samples were tested inparallel through all new designed assays. The forward primer/detectorsequence remained unchanged. The same PCR conditions were used as setout above.

TABLE 5  Newly designed reverse primer sequences Primer nameReverse primer sequence PMR103 5′-TAAAAA

CCTACAAAACCACT

-3′ (SEQ ID NO: 8) PMR104 5′-AAAAA

CCTACAAAACCACT

A-3′ (SEQ ID NO: 9) PMR105 5′-CT

AAACTACCAC

TCC

A-3′ (SEQ ID NO: 3) PMR106 5′-CTCGAAACTACCACCGTCCCG-3′ (SEQ ID NO: 10)PMR108 5′-ACTCCGCACTCTTCCGAAAACGA-3′ (SEQ ID NO: 11) PMR1115′-AACTCCGCACTCTTCCGAAAACG-3′ (SEQ ID NO: 12)

Table 6 summarizes the Ct values obtained for the 6 new primer deigns. Ahigh variation in NTC performance was observed depending on the chosenprimer set. The best NTC performance was obtained for PMR105: 13 out of14 tested water controls resulted in Ct values above 40.

The applied test run validation criteria for the amplifluor assay aredefined, in such a way that a “valid” Ct value has to be <40 (based on atheoretical definition: if there was one copy in the PCR reaction to beamplified it would take 40 cycles at a PCR efficiency of 100%). CLvalues above 40 are considered as ‘invalid’. Accordingly, a signal isconsidered to be negative in real-time MSP when it is classified as“undetermined” or when its Ct value is above 40.

TABLE 6 NTC performance of new MGMT designs Ct Ct Ct Ct Ct Ct SampleTask PMR103 PMR104 PMR105 PMR106 PMR108 PMR111 1 NTC 40.86 37.17 49.4740.10 38.99 39.44 2 NTC 41.35 35.32 40.60 39.38 39.96 37.88 3 NTC 42.4635.51 54.59 41.53 38.54 40.56 4 NTC 38.62 35.83 43.83 39.31 40.30 38.785 NTC 38.65 35.59 40.91 38.66 38.08 38.28 6 NTC 39.07 35.34 40.49 38.0539.29 40.43 7 NTC 39.29 35.46 44.41 37.92 38.61 39.54 8 NTC 34.95 37.7041.69 37.17 38.85 39.53 9 NTC 37.61 36.42 45.74 39.04 39.65 38.73 10 NTC38.91 36.37 40.19 37.97 38.29 39.28 11 NTC 39.78 37.63 37.98 40.51 39.4139.52 12 NTC 38.80 37.09 43.22 38.23 39.30 38.74 13 NTC 36.34 33.9445.92 38.70 38.48 38.38 14 NTC 40.93 36.53 40.18 42.99 38.91 39.40

Temperature Gradient

PMR105 was retained for further analysis. A temperature gradient wasperformed to investigate whether higher reading temperatures couldimprove the NTC performance.

The same reaction mixture and cycling conditions were used as set outabove.

5 different reading temperatures were tested on the I-cycler real-timeinstrument from BioRad: 62° C., 64.1° C., 66° C., 68.6° C. and 70.5° C.

Eight water controls were run per temperature, in addition a serialdilution of cloned MGMT M promoter (2×10̂6, 2×10̂4, 2×10̂2 and 2×10̂1 copynumbers) was tested to assess how higher reading temperatures influencethe sensitivity and PCR efficiency of the targeted assay. Correspondingresults are summarized in Table 7.

TABLE 7 Assay (including MGMT serial dilution and NTC) performance usinghigher reading temperatures Reading temperatures and PCR efficiency 62°C. 64.1° C. 66° C. 68.6° C. 70.5° C. Quantity 96% 98% 102% 101% 93%2000000 17.80 17.80 17.90 18.90 27.30 20000 24.80 24.80 24.70 25.5033.80 200 31.70 31.80 31.80 32.70 41.20 20 34.90 34.50 33.90 35.10 44.800 (NTC) 36.20 37.80 47.50 39.60 48.80 0 (NTC) 36.70 37.10 36.60 43.0051.60 0 (NTC) 36.90 38.10 40.80 38.40 40.80 0 (NTC) 36.90 37.50 38.2040.00 42.40 0 (NTC) 38.20 37.80 37.60 37.80 41.60 0 (NTC) 38.20 37.1037.60 42.20 40.70 0 (NTC) 39.30 38.60 36.70 39.70 40.70 0 (NTC) 45.2037.30 37.80 43.00 39.00

Based on Table 7, it can be concluded that higher reading temperaturesdo improve the NTC performance but at the same time reduces the assaysensitivity.

Reading temperatures 62° C. and 68.6° C. were retained for furtheranalysis. Both temperatures were tested on an initial set of 13 clinicalsamples to evaluate the sensitivity issue.

Clinical Results

13 glioma samples were run through the MGMT Amplifluor assay (PMR105)testing 2 different reading temperatures: 62° C. and 68.6° C.

Final primer concentrations in the Amplifluor reaction mix were 100 nMfor both forward primer/detector and reverse primer. 12.5 μl of iTaq™Supermix with Rox (BioRad, 2×buffer) were used per PCR reaction. Thetotal volume per reaction, including 5 μl of bisulfite treated DNA, was25 μl. The following thermal profile was used on the ABI 7900 HT SDSinstrument: Stage1: 50° C. for 2 min, Stage2: 95° C. for 10 min, Stage3:95° C. for 15 sec, 62° C. (alternatively 68.6° C.) for 1 min(=plateau-data collection) for 45 repeats. A serial dilution (2×10̂6—20copies) of MGMT M promoter was included to determine the copy numbers ofthe unknown samples by interpolation of their Ct values to the standardcurve. Results are summarized in Table 8.

TABLE 8 Performance of clinical samples using different readingtemperatures Copies MGMT Copies MGMT Sample no (PCR105) 62° C. (PMR105)68.6° C. 84 45 1 253 21 0 262 330 68 269 5 0 284 742 76 293 3 0 302 4 0309 48 0 322 4 0 403 948 262 433 2 1 460 277 28 532 9 0

Reading at 68.6° C. results in a considerable loss of sensitivitycompared to a reading temperature of 62° C.

It can be concluded that best workable results for the MGMT Amplifluorassay are obtained using the reverse primer design PMR105 in combinationwith a reading temperature of 62° C.

EXAMPLE 3 Additional Genes Tested Through Amplifluor

We developed a direct real-time fluorescence based methylation-specificPCR assay (real-time MSP assay) to define the methylation status of theMGMT promoter. Briefly, genomic DNA is deaminated using sodiumbisulphite after isolation. 5-Methyl Cytosine is refractory to thischemical modification. Unmethylated Cytosine quantitatively turns intoUracil during this process. After amplification of the DNA sequencesusing methylation specific primers, the detection of the amplified DNAsequences is carried out using the amplifluor technology. Themodification for the amplifluor primers is 5′ FAM internal Dabcyl.

The quantitation process depends on fluorescent light which is emittedonly when the detector is bound to its complementary sequence.

Analyte quantitations for several markers additional to MGMT weresuccessfully performed using this technology. These consisted ofparallel amplification/quantification processes using specific primerand primer/detector pairs for each analyte using the Amplifluor® assayformat on an ABI Prism® 7900HT instrument (Applied Biosystems).

The final primer concentrations in the reaction mix were 100 nM for bothforward primer/detector and reverse primer. 12.5 μl of iTaq™ Supermixwith Rox (BioRad, 2×buffer) were used per PCR reaction. The total volumeper reaction, including 5 μl of modified template DNA, was 25 μl. TheABI 7900HT SDS instrument was started 10 min before use, allowing theheated cover to reach 105° C. The following thermal profile was used:Stage1: 50° C. for 2 min, Stage2: 95° C. for 10 min, Stage3: 95° C. for15 sec, 62° C. for 1 min (=plateau-data collection) for 45 repeats.

Plasmid material, used as standard curve was generated as follows: thepromoter sequence as defined by the primers is PCR amplified and cloned(using suitable isolated and bisulphite modified cell line DNA). Thesequence is verified by sequencing and compared to the publishedpromoter sequence.

A standard curve (2×10⁶—20 copies) was included to determine copynumbers of unknown samples by in of their Ct values to the standardcurve. β-Actin was used as a reference gene in the assay.

BRCA1

Primer and Detector Sequences

A BRCA1 amplifluor assay was designed to evaluate the methylation statusof the BRCA1 gene in breast cancer patients. Primer and detectorsequences are detailed in Table 9.

TABLE 9 Primer and amplifluor detector sequences BRCA1 Name SequenceBRCA1 forward 5′-AGCGATGCGTTCGAGCATCGCUTCGTGGTAA detector CGGAAAAGCGC-3′(SEQ ID NO: 1 plus SEQ  ID NO: 6) BRCA1 reverse5′-AAATCTCAACGAACTCACGCCG-3′ detector (SEQ ID NO: 13)

Performance Standard Curve

A serial dilution of BRCA1 plasmid material (2×10⁶ to 2×10¹ copies/5 μl)was loaded in duplicate using the above specified primer and Amplifluordetector sequences. Results were generated (see Table 10) using the SDS2.2 software (Applied Biosystems), exported as Ct values (cycle numberat which the amplification curves cross the threshold value, setautomatically by the software). Good performance of the standard curvewas obtained: slope of −3.4147, corresponding to a PCR efficiency of 96%and R² value of 0.9999.

TABLE 10 performance BRCA1 standard curve Quantity Average Standards Ct₁Ct₂ Ct ΔCt 2000000 18.39 18.33 18.36 0.06 200000 22.30 22.15 22.22 0.1520000 26.19 26.17 26.18 0.03 2000 30.12 30.24 30.18 0.11 200 33.97 33.9233.95 0.04 20 37.92 37.90 37.91 0.02 NTC 42.30 41.55 41.93

Clinical Samples

The BRCA1 methylation status was investigated for 40 breast tumorsamples and 10 normal breast samples. The Ct values were used tocalculate the copy numbers for each sample based on a linear regressionof the standard curve values. The BRCA1 copy numbers were divided by theβ-Actin copy numbers and multiplied by 1000 for convenient handling;results were referred to as the ratio value (see Table 11). Invalidresults are due to β-Actin copy numbers below 200 in the respectivesamples. The results clearly show that methylated BRCA1 can bedistinguished from unmethylated BRCA1 with present assay set up.

TABLE 11 Preliminary results BRCA1 amplifluor assay on clinical materialRatio BRCA1/b-actin METHYLATION Sample Ct Copies (copies) × 1000 STATUSTumor 1 31.37 39.06 37.44 METHYLATED Tumor 2 31.21 43.55 75.26METHYLATED Tumor 3 31.67 31.98 59.44 METHYLATED Tumor 4 30.63 64.1487.40 METHYLATED Tumor 5 30.80 57.12 109.45 METHYLATED Tumor 6 33.996.74 51.38 INVALID Tumor 7 30.45 72.42 169.91 METHYLATED Tumor 8 30.5965.73 113.23 METHYLATED Tumor 9 26.47 1047.30 1184.29 METHYLATED Tumor10 27.70 458.09 184.54 METHYLATED Tumor 11 29.75 115.63 193.56METHYLATED Tumor 12 36.27 1.45 30.51 INVALID Tumor 13 31.47 36.53 101.88METHYLATED Tumor 14 32.35 20.21 55.18 METHYLATED Tumor 15 30.25 82.9327.54 METHYLATED Tumor 16 30.47 71.36 25.67 METHYLATED Tumor 17 30.4273.70 37.62 METHYLATED Tumor 18 32.34 20.40 25.83 METHYLATED Tumor 1931.40 38.22 50.15 METHYLATED Tumor 20 31.63 32.71 54.27 METHYLATED Tumor21 30.55 67.88 70.81 METHYLATED Tumor 22 31.67 31.99 36.60 METHYLATEDTumor 23 31.43 37.46 108.74 METHYLATED Tumor 24 31.71 31.14 92.78METHYLATED Tumor 25 34.85 3.77 49.66 INVALID Tumor 26 31.50 35.70 78.28METHYLATED Tumor 27 31.03 49.09 135.65 METHYLATED Tumor 28 31.46 36.6278.37 METHYLATED Tumor 29 26.66 922.65 1098.17 METHYLATED Tumor 30 30.7260.18 19.55 METHYLATED Tumor 31 30.06 94.17 62.02 METHYLATED Tumor 3232.09 24.08 62.53 METHYLATED Tumor 33 30.11 91.01 173.98 METHYLATEDTumor 34 34.40 5.10 116.39 INVALID Tumor 35 39.88 0.13 #DIV/0! INVALIDTumor 36 31.60 33.39 148.31 METHYLATED Tumor 37 34.17 5.95 27.68UNMETHYLATED Tumor 38 38.14 0.42 47.87 INVALID Tumor 39 39.34 0.19176.66 INVALID Tumor 40 Undeter- 0.00 0.00 INVALID mined Normal 1 37.650.58 21.37 INVALID Normal 2 36.84 0.99 14.18 INVALID Normal 3 38.89 0.257.78 INVALID Normal 4 38.19 0.40 4.20 INVALID Normal 5 37.66 0.57 2.11UNMETHYLATED Normal 6 40.28 0.00 0.00 UNMETHYLATED Normal 7 35.92 1.8423.08 INVALID Normal 8 38.07 0.43 17.15 INVALID Normal 9 41.77 0.00 0.00INVALID Normal 10 41.41 0.00 0.00 UNMETHYLATED positive 24.12 5060.951143.85 METHYLATED control negative 36.79 1.03 2.58 UNMETHYLATED control

WRN

Primer and Detector Sequences

Different WRN amplifluor assays were designed to evaluate themethylation status of the WRN gene in colorectal cancer patients. Primerand detector sequences are detailed in Table 12.

TABLE 12 Primer and amplifluor detector sequences WRN Name SequenceWRN_NOR1 5′-AGCGATGCGTTCGAGCATCGCUGTTCGTATT forwardGTTTTTCGTCGGAGTAGTC-3′  (SEQ ID  detector NO: 1 plus SEQ ID NO: 14)WRN_NOR1 5′-CGCAACGACCGCAAAAAAAACG-3′ reverse (SEQ ID NO: 15) detectorWRN NOR1 5′-AGCGATGCGTTCGAGCATCGCUCGCAACGAC reverse CGCAAAAAAAACG-3′(SEQ ID  detector NO: 1 plus SEQ ID NO: 15) WRN_NOR1GTTCGTATTGTTTTTCGTCGGAGTAGTC-3′ forward (SEQ ID NO: 14) primer WRN_NOR25′-AGCGATGCGTTCGAGCATCGCUCCGACAATA reverse ACTAAAACCCCG-3′ (SEQ IDdetector NO: 1 plus SEQ ID NO: 16) WRN_NOR25′-GGGTGTTGAGAATAATCGTAGAC-3′ forward (SEQ ID NO: 17) primer WRN_NOR45′-AGCGATGCGTTCGAGCATCGCUTAATATAAA reverse TACCCGCCGACT-3′ (SEQ IDdetector NO: 1 plus SEQ ID NO: 18) WRN_NOR4 GTTTTGTTCGCGTTTTTCGTA-3′forward (SEQ ID NO: 19) primer

Performance Standard Curve

A serial dilution of WRN plasmid material (2×10⁶ to 2×10¹ copies/5 μl)was loaded in duplicate using the above specified primer and Amplifluordetector sequences using an optimized thermal profile: Stage1: 50° C.for 2 min, Stage2: 95° C. for 10 min, Stage3: 95° C. for 15 sec, for 61°C. for 30 sec, 61° C. for 30 sec (=plateau-data collection) for 45repeats. Different primer combinations were assessed for WRN. Resultswere generated (see: Table 15) using the SDS 2.2 software (AppliedBiosystems), exported as Ct values (cycle number at which theamplification curves cross the threshold value, set automatically by thesoftware). Good performance of the standard curve was obtained for allassays.

TABLE 13 performance WRN standard curve using different primercombinations Quantity Average Ct Average Ct Average Ct Average CtStandards WRN_NOR1F WRN_NOR1R WRN_NOR2R WRN_NOR4R 2000000 14.88 15.8214.41 16.83 200000 18.70 19.90 17.84 20.62 20000 22.56 23.98 21.58 24.202000 26.13 28.06 25.37 27.80 200 30.22 32.17 29.14 31.42 20 33.85 35.6833.19 34.50 NTC Undetermined Undetermined Undetermined Undetermined PCRefficiency (%) 83 78 84 91 R² 0.9998 0.9996 0.9995 0.9992 (NTC =no-template control)

Clinical Samples

The WRN methylation status was investigated for 56 colorectal cancersamples and 39 normal samples. The Ct values were used to calculate thecopy numbers for each sample based on a linear regression of thestandard curve values. The WRN copy numbers were divided by the β-Actincopy numbers and multiplied by 1000 for convenient handling; resultswere referred to as the ratio valve (see Table 14). Performancecharacteristic of the different WRN assays: WRN_NOR1F, WRN_NOR1R,WRN_NOR2R and WRN_NOR4R demonstrated a sensitivity of respectively 41%,49%, 30% and 50% with a specificity of 100% for the tested CRC.

TABLE 14 Preliminary results WRN amplifluor assay on clinical materialratio ratio ratio ratio WRN_NOR1F/b- WRN_NOR1R/b- WRN_NOR2R/b-WRN_NOR4R/b- Samples Group actin × 1000 actin × 1000 actin × 1000 actin× 1000 Cut_off 39.07 13.44 3.23 6.70 Specificity % 100 100 100 100Sensitivity % 41 49 30 50

The different WRN assays were tested on their compiementarities fordetecting CRC; the best combination was obtained with WRN_NOR1R andWRN_NOR4R. Results are detailed in Table 15.

TABLE 15 Complementary results WRN_NOR1R and WRN_NOR4R SpecificitySensitivity Group Methylated Unmethylated Invalid Valid % % WRN_NOR1R-normal 0 36 3 36 100 NOR4R cancer 32 17 7 49 65

PTEN

Primer and Detector Sequences

Different PTEN amplifluor assays were designed to evaluate themethylation status of the PTEN gene in cancerous patients. Sequencedetails can be found in Table 16.

TABLE 16 Primer and amplifiuor detector sequences PTEN Name SequencePTEN 11 5′-AGCGATGCGTTCGAGCATCGCUGTGTTTACGTTAGT forward ACGTTCGGT-3′(SEQ ID NO: 1 plus detector SEQ ID NO: 20) PTEN5′-TCATCCGACTCCCTTACAACG-3′ reverse (SEQ ID NO: 21) primer PTEN 125′-AGCGATGCGTTCGAGCATCGCUTAGTTTTGGGTGCG forward AGCGTAG-3′(SEQ ID NO: 1 plus detector SEQ ID NO: 22) PTEN 125′-GCGTTACTACAAAAACCGCAA-3′ reverse (SEQ ID NO: 23) primer PTEN 65′-AGCGATGCGTTCGAGCATCGCUTGGTATATTTAGGG forward ATTCGGGTC-3′(SEQ ID NO: 1 plus primer SEQ ID NO: 24) PTEN 65′-AACGAATAATCCTCCGAACG-3′ reverse (SEQ ID NO: 25) primer

Performance Standard Curve

A serial dilution of PTEN alternative standard curve material (2×10⁶ to2×10¹ copies/5 μl) was loaded in duplicate using the above specifiedprimer and Amplifluor detector sequences. Results were generated usingthe SDS 2.2 software (Applied Biosystems), exported as Ct values (cyclenumber at which the amplification curves cross the threshold value, setautomatically by the software). Good performance of the standard curvewas obtained. Results are summarized is Table 17.

TABLE 17 Summary of slopes and PCR efficiencies PTEN Name Slope R²Efficiency PTEN 11 standard curve material 3.6769 0.9996 87% PTEN 12standard curve material 3.5803 0.9999 90% PTEN 6 standard curve material3.3864 0.9974 97%

NDRG4

Primer and Detector Sequences

Different NDRG4 amplifluor assays were tested to evaluate themethylation status of the NDRG4 gene in colorectal cancer patients.Especially short amplifluor assays were designed to favour the detectionof low-molecular-weight DNA, often present in plasma/serum samples inthe form of fragmented DNA. Primer and detector sequences are detailedin Table 18.

TABLE 18 Primer and amplifluor detector sequences NDRG4  5′ to 3′Sequences Detector Modifications:  Name 5′ FAM and internal dUdabcylNDRG4_66304_ AGCGATGCGTTCGAGCATCGCUTTCGGTGAATTT S_AMP forwardTAGGAGGC (SEQ ID NO: 1 plus detector SEQ ID NO: 26) NDRG4_66304_TCGAACGACGAACACGAAA (SEQ ID NO: 27) AS reverse primer NDRG4_72006_AGCGATGCGTTCGAGCATCGCUCGTTCGGGATTA S_AMP forwardGTTTTAGGTTC (SEQ ID NO: 1 plus detector SEQ ID NO: 28) NDRG4_72006_AATTTAACGAATATAAACGCTCG AS reverse (SEQ ID NO: 29) primer NDRG4_72007_AGCGATGCGTTCGAGCATCGCUGGTATTTTAGTC S_AMP forwardCGTAGAAGGC (SEQ ID NO: 1 plus detector (SEQ ID NO: 30) NDRG4_72007_ACTAATCCCGAACGAACCG  AS reverse (SEQ ID NO: 31) primer NDRG4_72008_CGTTCGGAGTTCGTTTTAATTAC S_AMP forward (SEQ ID NO: 32) detectorNDRG4_72008_ AGCGATGCGTTCGAGCATCGCUCTACTCACAAAT AS_AMP ReverseACCGCCCG (SEQ ID NO: 1 plus detector SEQ ID NO: 33)

Performance Standard Curve

A serial dilution of NDRG4 alternative standard curve material (9.6×10⁶to 9.6×10¹ copies/5 μl) was loaded in duplicate using the abovespecified primer and Amplifluor detector sequences.

Alternative standard curve material was generated as follow: thepromoter sequence as defined by the primers was PCR amplified, loaded ongel to check its specificity, subsequently purified and quantified usingthe Picogreen® dsDNA quantitation kit (Molecular Probes, #P7589)according to the manufacturer's recommendations.

2.4 μl of standard curve dilution was added to a final 12 μl PCRreaction mix volume containing: 6 μl of QuantiTect Multiplex Master Mix(Qiagen, 2×buffer) and final primer concentrations of 100 nM for bothprimer and detector sequences.

Cycling conditions for each NDRG4 design were 95° C. for 15 min;followed by 45 cycles of 94° C. for 15 sec, 57° C. for 30 sec [62° C.for NDRG4_(—)72008] and 57° C. for 30 sec [62° C. for NDRG4_(—)72008](=plateau-data collection).

Different primer combinations were assessed for NDRG4. Results weregenerated (see Table 19) using the SDS 2.2.2 software from AppliedBiosystems with automatic baseline and threshold settings. Goodperformance of the standard curve was obtained for all assays.

TABLE 19 performance NDRG4 standard curve using different primercombinations Amplicon Assay name length Slope R² Efficiency NTCNDRG4_66304 107 3.3800 0.9994 97.6% negative NDRG4_72006 85 3.64020.9987 88.2% negative NDRG4_72007 86 3.4834 0.9998 93.7% negativeNDRG4_72008 92 3.2681 0.9999 102.3% negative

Clinical Samples

1) Evaluation sensitivity of NDRG4 Amplifluor Assays Using DilutedPlasma Samples from Patients Diagnosed with CRC

10 plasma samples from colorectal cancer patients (stage IV and III)were provided by Signature Diagnostics. Each sample was split in 3 anddiluted with plasma from healthy volunteers (control) according tofollowing dilution scheme:

Condition A: 2 ml CRC plasma+2 ml control plasma

Condition B: 1 ml CRC plasma+3 ml control plasma

Condition C: 2 ml control plasma (negative control)

Genomic DNA was extracted using a standard method (phenol/chloroform),followed by sodium bisulfite treatment (BT) using the EZ DNA Methylationkit from Zyme Research. The chemically treated DNA was used as templatefor real-time MSP. Methylation levels of the NDRG4 gene promoter weredetermined by real-time MSP using specific primers and probes for themolecular beacon format NDRG4_(—)1a (details of this assay havepreviously been provided in International Publication WO08/084219) andamplifluor format: NDRG4_(—)72007 and NDRG4_(—)72008.

Recovered copy numbers were calculated based on a linear regression ofthe standard curve and compared for each different condition.

TABLE 20 copy number recovery NDRG4 beacon format versus amplifluorformat copies Copies Copies Condition NDRG4_1a NDRG4_72007 NDRG4_72008samples (beacon) (amplifluor) (amplifluor) Sample 1-A 2.12 31.55 23.43Sample 2-A 0.00 0.00 0.92 Sample 3-A 0.00 0.00 0.00 Sample 4-A 33.18265.55 107.16 Sample 5-A 0.00 3.31 0.00 Sample 6-A 19.55 369.07 675.73Sample 7-A 2.64 12.00 23.23 Sample 8-A 0.00 0.00 54.09 Sample 9-A 0.000.00 0.00 Sample 10-A 0.00 0.00 2.03 Sample 1-B 2.97 13.84 39.15 Sample2-B 0.00 0.00 0.89 Sample 3-B 0.00 0.00 0.00 Sample 4-B 42.13 321.5176.25 Sample 5-B 0.00 0.00 1.40 Sample 6-B N/A N/A N/A Sample 7-B 0.005.53 0.00 Sample 8-B N/A N/A N/A Sample 9-B 0.00 0.00 2.16 Sample 10-B0.00 0.00 0.00 Sample 1-C 0.00 0.00 10.05 Sample 2-C 3.68 0.00 0.00Sample 3-C 0.00 0.00 0.00 Sample 4-C 0.00 0.00 1.00 Sample 5-C 0.00 0.000.00 Sample 6-C 0.00 0.00 0.00 Sample 7-C 0.00 0.00 4.58 Sample 8-C 0.000.00 0.00 Sample 9-C 0.00 0.00 5.64 Sample 10-C 0.00 0.00 0.98 poscontrol 5671.29 23164.81 18937.12 neg control 0.00 0.00 0.00

Results are presented in Table 20. Higher copy numbers are obtainedusing the amplifluor format. Performance characteristics of thedifferent NDRG4 assays: NDRG4_(—)1a (beacon), NDRG4_(—)72007(amplifluor), and NDRG4_(—)72008 (amplifluor) demonstrated a sensitivityof respectively 20%, 50%, and 50% with a specificity of 100% for thetested CRC diluted plasma samples. These preliminary data indicate thata short amplifluor assay could provide a better alternative than beaconassay (technically difficult to shorten the assay) for CRC methylationdetection in plasma DNA samples (high DNA fragmentation nature).

2) NDRG4_(—)72007 Amplifluor Assay and NDRG4_(—)1a Beacon Assay Testedon Tissue Samples

The NDRG4 methylation status was further investigated in 88 CRC FFPE(formalin fixed paraffin embedded) samples and 10 normal FFPE samples.

Formalin Fixed paraffin embedded samples were first de-paraffinized in750 μl xylene for 2 h. Then, 250 μl of 70% ethanol was added beforecentrifugation at 13000 rpm for 15 min. The supernatant was removed andthe samples were air dried at room temperature.

Genomic DNA was extracted using a standard method (phenol/chloroform),followed by sodium bisulfite treatment (BT) using the EZ DNA.Methylation kit from Zymo Research.

1.5 μl of chemically treated DNA was used as template for real-time MSP.Methylation levels of the NDRG4 gene promoter were determined byreal-time MSP using specific primers and probes for the molecular beaconformat: NDRG4_(—)1a and amplifluor format: NDRG4_(—)72007. Severalannealing temperatures were investigated (57° C., 62° C. and 80.5° C.)to further optimize the NDRG4_(—)72007 amplifluor assay.

The results presented in Table 21 clearly indicate that present NDRG4assay set-ups, discriminate between cancers and non-cancers.

TABLE 21 Performance characteristics for NDRG4_1a and NDRG4_72007NDRG4_72007 NDRG4_72007 NDRG4_72007 NDRG4_1a 57° C. 62° C. 80.5° C.(beacon) (amplifluor) (amplifluor) (amplifluor) No cut-off appliedSensitivity (%) 57 83 71 56 Specificity (%) 93 64 95 100 Cut-off appliedto obtain 100% specificity Cut-off ratio 2.26 80 390 / Sensitivity (%)55 78 63 /

EXAMPLE 4 Reproducibility of the MGMT Amplifluor Assay

To test the reproducibility of the MGMT amplifluor assay as described inexample 1, 75 available clinical samples were processed twice throughreal-time MSP: once at OncoMethylome Sciences (ONCO) and once atLaboratory Corporation of America® Holdings (LabCorp®). Bothlaboratories obtained comparable results.

Materials and Methods

Sample Preparation

75 formalin-fixed, paraffin-embedded (FFPE) glioma tissue samples wereavailable for testing through the MGMT amplifluor real-time MSP. Foreach glioma tumor sample, 4 10 μm consecutive sections were prepared onglass slides. The prepared sample sections were then divided between the2 sites and processed in parallel (including sample preparation)according to each laboratory's respective protocols (both real-timeMSP). LabCorp® was blinded to the results obtained by ONCO.

Real-Time MSP Protocol Followed by ONCO

DNA isolation, modification and analyte quantitations were conducted aspreviously described in example 1;

amplification conditions were subject to the following modifications:

-   -   iTaq™ Supermix with Rox (BioRad, 2×buffer) was replaced by        Quantitect buffer (Qiagen, 2×buffer)    -   Stage3 of the thermal profile was further optimized: 95° C. for        15 sec, 62° C. for 30 sec and 62° C. for 30 sec (=plateau-data        collection)

Technical test run validation criteria used to classify samples asmethylated, non-methylated or invalid are described in Table 22. Acutoff of 8 was used for classification of non-methylated and methylatedMGMT.

TABLE 22 MGMT validation criteria Results copy# (mMGMT) copy# (β-actin)Ratio × 1000 Methylated MGMT ≧10 ≧10 ≧8 Non-Methylated ≧10 ≧10 <8 MGMT<10 ≧1250 / Invalid <10 <1250 / any <10 /

Real-Time MSP Protocol Followed by LabCorp®

Genomic DNA Extraction, Purification and Modification was According tothe In-House Procedure Used at LabCorp®

Real-time MSP procedure was conducted similarly to the protocol followedby ONCO.

Technical test run validation criteria are described in Table 22.

Results

Several patient samples were processed in parallel in independentlaboratories to confirm the reproducibility of the MGMT assay. Sampleprocessing was performed according to each laboratory's respectiveprotocol. In total, 75 glioma (glioblastoma) samples, blinded toLabCorp®, were processed through the MGMT assay as specified inmaterials and methods to demonstrate similar results obtained at ONCO.Results obtained in each laboratory are indicated in Table 23.

TABLE 23 Results 75 glioma samples ONCO versus LabCorp ® Sample # ONCOLabCorp ® 1 Non-methylated Non-methylated 2 Non-methylatedNon-methylated 3 Methylated Methylated 4 Non-methylated Non-methylated 5Non-methylated Non-methylated 6 Methylated Methylated 7 Non-methylatedNon-methylated 8 Methylated Methylated 9 Methylated Methylated 10Non-methylated Non-methylated 11 Methylated Methylated 12 Non-methylatedNon-methylated 13 Non-methylated Non-methylated 14 Non-methylatedNon-methylated 15 Non-methylated Non-methylated 16 Non-methylatedNon-methylated 17 Non-methylated Non-methylated 18 Methylated Methylated19 Methylated Methylated 20 Non-methylated Non-methylated 21Non-methylated Non-methylated 22 Methylated Methylated 23 Non-methylatedNon-methylated 24 Methylated Methylated 25 Non-methylated Non-methylated26 Non-methylated Non-methylated 27 Methylated Methylated 28Non-methylated Non-methylated 29 Non-methylated Non-methylated 30Non-methylated Non-methylated 31 Methylated Methylated 32 MethylatedMethylated 33 Non-methylated Invalid 34 Non-methylated Non-methylated 35Methylated Methylated 36 Non-methylated Non-methylated 37 Non-methylatedNon-methylated 38 Non-methylated Non-methylated 39 Non-methylatedNon-methylated 40 Non-methylated Invalid 41 Non-methylated Invalid 42Non-methylated Invalid 43 Non-methylated Invalid 44 Non-methylatedInvalid 45 Non-methylated Non-methylated 46 Non-methylatedNon-methylated 47 Non-methylated Invalid 48 Non-methylated Invalid 49Non-methylated Invalid 50 Non-methylated Non-methylated 51Non-methylated Invalid 52 Non-methylated Non-methylated 53 MethylatedMethylated 54 Non-methylated Non-methylated 55 Non-methylatedNon-methylated 56 Non-methylated Non-methylated 57 Non-methylatedNon-methylated 58 Methylated Methylated 59 Methylated Invalid 60Methylated Methylated 61 Non-methylated Non-methylated 62 MethylatedMethylated 63 Non-methylated Non-methylated 64 Non-methylated Invalid 65Non-methylated Non-methylated 66 Methylated Methylated 67 Non-methylatedNon-methylated 68 Methylated Methylated 69 Invalid Invalid 70Non-methylated Non-methylated 71 Methylated Methylated 72 Non-methylatedNon-methylated 73 Methylated Methylated 74 Methylated Methylated 75Methylated Methylated

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope or theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A real-time method of detecting the presence and/or amount of amethylated or unmethylated gene of interest in a DNA-containing sample,comprising: (a) contacting the DNA-containing sample with a reagentwhich selectively modifies unmethylated cytosine residues in the DNA toproduce detectable modified residues but which does not modifymethylated cytosine residues (b) amplifying at least a portion of themethylated or unmethylated gene of interest using at least one primerpair, at least one primer of which is designed to bind only to thesequence of methylated or unmethylated DNA following treatment with thereagent, wherein at least one primer in the primer pair is a primercontaining a stem loop structure carrying a donor and an acceptor moietyof a molecular energy transfer pair arranged such that in the absence ofamplification, the acceptor moiety quenches fluorescence emitted by thedonor moiety upon excitation and during amplification, the stem loopstructure is disrupted so as to separate the donor and acceptor moietiessufficiently to produce a detectable fluorescence signal which isdetected in real-time to provide an indication of the gene copy numberof the methylated or unmethylated gene of interest (c) quantifying theresults of the real-time detection against a standard curve for themethylated or unmethylated gene of interest to produce an output of genecopy number; characterised in that the amplification is considered validwhere the cycle threshold value is less than
 40. 2. The method of claim1 further characterised in amplifying at least a portion of a referencegene using at least one primer pair, wherein at least one printer in theprimer pair is a primer containing a stem loop structure carrying adonor and an acceptor moiety of a molecular energy transfer pairarranged such that in the absence of amplification, the acceptor moietyquenches fluorescence emitted by the donor moiety upon excitation andduring amplification, the stem loop structure is disrupted so as toseparate the donor and acceptor moieties sufficiently to produce adetectable fluorescence signal which is detected in real-time to providean indication of the gene copy number of the reference gene.
 3. Themethod of claim 2 further characterised in that the results of thereal-time detection are quantified against both a standard curve for themethylated or unmethylated gene of interest and reference generespectively to produce an output of gene copy number and the resultsare normalised by dividing the gene copy number of the methylated orunmethylated gene of interest by the gene copy number of the referencegene. 4.-5. (canceled)
 6. The method of claim 1 further characterised inthat the amplification is considered valid where: (a) the slope of thestandard curve for the methylated or unmethylated gene of interest andoptionally the reference gene is at least −4, indicating anamplification efficiency of at least 77%, (b) the coefficient ofdetermination (R2) for at least four data points on the standard curveor curves is above 0.99; (c) in a parallel reaction using the samereagents, there is no amplification of a sample containing no DNA at thecycle threshold value of less than 40; (d) in a parallel reaction usingthe same reagents, there is detectable amplification of a positivecontrol sample known to contain the gene of interest in methylated orunmethylated form at the cycle threshold value of less than 40; or (e)in a parallel reaction using the same reagents, there is no detectableamplification of a negative control sample known to contain the gene ofinterest in unmethylated or methylated form at the cycle threshold valueof less than
 40. 7.-10. (canceled)
 11. The method of claim 1, whereinthe methylated gene of interest is selected from MGMT, WRN, BRCA1, PTENand NDRG4. 12.-17. (canceled)
 18. The method of claim 11 wherein thegene of interest is MGMT and the positive control sample known tocontain methylated MGMT is derived from SW48 cells and/or wherein thegene of interest is MGMT and the negative control sample known tocontain unmethylated MGMT is derived from HT29 cells.
 19. (canceled) 20.The method of claim 1, wherein the at least one primer pair foramplification of the methylated or unmethylated gene ofinterest/reference gene is used in the amplification at a concentrationof approximately 50 to 150 nM or at a concentration of approximately 100nM.
 21. (canceled)
 22. The method of claim 1, wherein the amplifying iscarried out using the polymerase chain reaction and the data collectionstep is carried out at 62° C.
 23. (canceled)
 24. The method of claim 22,wherein the thermal profiling of the polymerase chain reaction comprisesapproximately 45 repeats of the cycle: (a) 50° C. for 2 minutes (b) 95°C. for 10 minutes (c) 95° C. for 15 seconds (d) 62° C. for 1 minute25-26. (canceled)
 27. The method of claim 1, wherein the at least oneprimer pair for amplification of the methylated or unmethylated gene ofinterest/reference gene produces an amplification product of betweenapproximately 50 and 250 bp or produces an amplification product ofbetween approximately 100 and 200 bp.
 28. (canceled)
 29. The method ofclaim 2, wherein amplification of the gene of interest and referencegene is carried out simultaneously in the same reaction.
 30. The methodof claim 1, wherein the total reaction volume for the amplification stepis around 25 μl. 31.-43. (canceled)
 44. A primer pair for real timedetection of: (a) the methylated MGMT gene comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID NO: 2 and 3; (b) the methylated WRN gene comprising, consistingessentially of or consisting of the nucleotide sequences set forth asSEQ ID NO:14 and SEQ ID NO:15; SEQ ID NO:16 and SEQ ID NO:17; or SEQ IDNO:18 and SEQ ID NO:19; (c) the methylated PTEN gene comprising,consisting essentially of or consisting of the nucleotide sequences setforth as SEQ ID NO:20 and SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23;or SEQ ID NO:24 and SEQ ID NO:25; (d) the methylated NDRG4 genecomprising, consisting essentially of or consisting of the nucleotidesequences set forth as SEQ ID NO:26 and SEQ ID NO:27 with the provisothat at least one primer in the primer pair further comprises a stemloop structure carrying a donor and an acceptor moiety of a molecularenergy transfer pair arranged such that in the absence of amplification,the acceptor moiety quenches fluorescence emitted by the donor moietyupon excitation and during amplification, the stem loop structure isdisrupted so as to separate the donor and acceptor moieties sufficientlyto produce a detectable fluorescence signal, SEQ ID NO:28 and SEQ IDNO:29; SEQ ID NO:30 and SEQ ID NO:31; or SEQ ID NO:32 and SEQ ID NO:33;(e) the beta-actin gene comprising the nucleotide sequences set forthSEQ ID NO:4 and
 5. 45.-57. (canceled)
 58. A kit for real-time detectionof a methylated gene comprising at least one primer pair as claimed inclaim
 44. 59. A method of predicting the likelihood of successfultreatment of a cell proliferative disorder in a subject using analkylating chemotherapeutic agent comprising, in a DNA-containing sampleisolated from the subject, detecting the presence and/or amount ofmethylated MGMT in the sample by carrying out the method of claim 1,wherein the presence of methylated MGMT in the sample indicates that thelikelihood of successful treatment using the alkylating chemotherapeuticagent is higher than if no or lower levels of methylated MGMT isdetected. 60.-66. (canceled)